<?xml version="1.0" encoding="UTF-8"?>

<!--XBRL Document Created with the Workiva Platform-->
<!--Copyright 2026 Workiva-->
<!--r:01a0df8f-191b-7707-ba7e-58848fbd6369,g:d672f85f-1ab3-4ffc-8116-661c59023419-->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"/>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"/>
  <link:roleRef roleURI="http://www.vistagen.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFCHANGESINSTOCKHOLDERSEQUITY" xlink:type="simple" xlink:href="vtgn-20260930.xsd#CONDENSEDCONSOLIDATEDSTATEMENTSOFCHANGESINSTOCKHOLDERSEQUITY"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/CONDENSEDCONSOLIDATEDSTATEMENTSOFCHANGESINSTOCKHOLDERSEQUITY" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementLineItems_01a0df8f-191c-7564-9dc4-1c030304cbe9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementTable_01a0df8f-191c-770d-bd2b-5b1bf5c42aad" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_StatementLineItems_01a0df8f-191c-7564-9dc4-1c030304cbe9" xlink:to="loc_us-gaap_StatementTable_01a0df8f-191c-770d-bd2b-5b1bf5c42aad" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementEquityComponentsAxis_01a0df8f-191c-7322-a924-058a2c673487" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementEquityComponentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_01a0df8f-191c-770d-bd2b-5b1bf5c42aad" xlink:to="loc_us-gaap_StatementEquityComponentsAxis_01a0df8f-191c-7322-a924-058a2c673487" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7322-a924-058a2c673487_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_01a0df8f-191c-7322-a924-058a2c673487" xlink:to="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7322-a924-058a2c673487_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EquityComponentDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_StatementEquityComponentsAxis_01a0df8f-191c-7322-a924-058a2c673487" xlink:to="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CommonStockMember_01a0df8f-191c-7754-baa0-f2e799d553cc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CommonStockMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:to="loc_us-gaap_CommonStockMember_01a0df8f-191c-7754-baa0-f2e799d553cc" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdditionalPaidInCapitalMember_01a0df8f-191c-77ef-9f9f-daafc5734036" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdditionalPaidInCapitalMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:to="loc_us-gaap_AdditionalPaidInCapitalMember_01a0df8f-191c-77ef-9f9f-daafc5734036" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TreasuryStockCommonMember_01a0df8f-191c-7e1a-be5e-ae5229229425" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TreasuryStockCommonMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:to="loc_us-gaap_TreasuryStockCommonMember_01a0df8f-191c-7e1a-be5e-ae5229229425" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_01a0df8f-191c-7270-ba25-f63896d99fac" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:to="loc_us-gaap_AccumulatedOtherComprehensiveIncomeMember_01a0df8f-191c-7270-ba25-f63896d99fac" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RetainedEarningsMember_01a0df8f-191c-7e86-ba24-810ec5ce2732" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RetainedEarningsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EquityComponentDomain_01a0df8f-191c-7f69-ba8b-1067dd7d0c83" xlink:to="loc_us-gaap_RetainedEarningsMember_01a0df8f-191c-7e86-ba24-810ec5ce2732" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-191c-71d5-be01-b2b425066a2c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_StatementTable_01a0df8f-191c-770d-bd2b-5b1bf5c42aad" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-191c-71d5-be01-b2b425066a2c" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-191c-71d5-be01-b2b425066a2c_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-191c-71d5-be01-b2b425066a2c" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-191c-71d5-be01-b2b425066a2c_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-191c-745d-bcc7-26fe85bb3250" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-191c-71d5-be01-b2b425066a2c" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-191c-745d-bcc7-26fe85bb3250" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_OpenMarketSaleAgreementMember_01a0df8f-191c-7c22-a5f4-b8ffce92c6d1" xlink:href="vtgn-20260930.xsd#vtgn_OpenMarketSaleAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-191c-745d-bcc7-26fe85bb3250" xlink:to="loc_vtgn_OpenMarketSaleAgreementMember_01a0df8f-191c-7c22-a5f4-b8ffce92c6d1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_StatementLineItems_01a0df8f-191c-7564-9dc4-1c030304cbe9" xlink:to="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharesOutstanding_01a0df8f-191c-7c76-a2e1-aa67263627c2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_SharesOutstanding_01a0df8f-191c-7c76-a2e1-aa67263627c2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_01a0df8f-191c-7b43-96d9-60651c63acdb" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_StockholdersEquity_01a0df8f-191c-7b43-96d9-60651c63acdb" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_01a0df8f-191c-7fa1-ae75-08086304e735" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue_01a0df8f-191c-7fa1-ae75-08086304e735" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_01a0df8f-191c-7bd9-9ed1-6f6c28688660" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans_01a0df8f-191c-7bd9-9ed1-6f6c28688660" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan_01a0df8f-191c-71ce-8adf-01756a6e29ad" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan_01a0df8f-191c-71ce-8adf-01756a6e29ad" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OtherComprehensiveIncomeLossAvailableForSaleSecuritiesAdjustmentBeforeReclassificationAdjustmentsNetOfTax_01a0df8f-191c-721e-aa47-0ea18f60d5a0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OtherComprehensiveIncomeLossAvailableForSaleSecuritiesAdjustmentBeforeReclassificationAdjustmentsNetOfTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_OtherComprehensiveIncomeLossAvailableForSaleSecuritiesAdjustmentBeforeReclassificationAdjustmentsNetOfTax_01a0df8f-191c-721e-aa47-0ea18f60d5a0" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a0df8f-191c-754f-9db1-c85ccd173783" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a0df8f-191c-754f-9db1-c85ccd173783" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_01a0df8f-191c-7b69-b06e-8bf219ce2ba5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_StockIssuedDuringPeriodValueNewIssues_01a0df8f-191c-7b69-b06e-8bf219ce2ba5" xlink:type="arc" order="8"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_StockIssuedDuringPeriodSharesAndWarrantsNewIssues_01a1c115-f5a3-78f9-a1fa-2c2717350475" xlink:href="vtgn-20260930.xsd#vtgn_StockIssuedDuringPeriodSharesAndWarrantsNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_vtgn_StockIssuedDuringPeriodSharesAndWarrantsNewIssues_01a1c115-f5a3-78f9-a1fa-2c2717350475" xlink:type="arc" order="9"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_StockIssuedDuringPeriodValueSharesAndWarrantsNewIssues_01a1c115-f5a3-7ef6-811d-cb68932821bd" xlink:href="vtgn-20260930.xsd#vtgn_StockIssuedDuringPeriodValueSharesAndWarrantsNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_vtgn_StockIssuedDuringPeriodValueSharesAndWarrantsNewIssues_01a1c115-f5a3-7ef6-811d-cb68932821bd" xlink:type="arc" order="10"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_01a1b236-b925-7b62-bd92-2a24dd38d980" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_01a1b236-b925-7b62-bd92-2a24dd38d980" xlink:type="arc" order="11"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NetIncomeLoss_01a0df8f-191c-7055-8be9-478b9acf8bf8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NetIncomeLoss"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_IncreaseDecreaseInStockholdersEquityRollForward_01a0df8f-191c-7f14-b031-6af88844dcf9" xlink:to="loc_us-gaap_NetIncomeLoss_01a0df8f-191c-7055-8be9-478b9acf8bf8" xlink:type="arc" order="12"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharesOutstanding_01a0df8f-191c-7d76-bafa-df629275adc2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharesOutstanding"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockholdersEquity_01a0df8f-191c-7e8a-868a-93b095e43480" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockholdersEquity"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/FairValueMeasurementsCashCashEquivalentsandMarketableSecuritiesDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#FairValueMeasurementsCashCashEquivalentsandMarketableSecuritiesDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/FairValueMeasurementsCashCashEquivalentsandMarketableSecuritiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bde-8809-9f199369c09d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-7405-9c2c-6891956b9e4c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bde-8809-9f199369c09d" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-7405-9c2c-6891956b9e4c" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_01a0df8f-19d2-7b80-82f2-5e327456d819" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-7405-9c2c-6891956b9e4c" xlink:to="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_01a0df8f-19d2-7b80-82f2-5e327456d819" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-7b80-82f2-5e327456d819_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_01a0df8f-19d2-7b80-82f2-5e327456d819" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-7b80-82f2-5e327456d819_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-79cf-97c3-e163f4064c10" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByFairValueHierarchyLevelAxis_01a0df8f-19d2-7b80-82f2-5e327456d819" xlink:to="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-79cf-97c3-e163f4064c10" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel1Member_01a0df8f-19d2-7697-b21f-6b589e0797cf" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel1Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-79cf-97c3-e163f4064c10" xlink:to="loc_us-gaap_FairValueInputsLevel1Member_01a0df8f-19d2-7697-b21f-6b589e0797cf" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel2Member_01a0df8f-19d2-7ce7-b9ca-045f67a7215c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel2Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-79cf-97c3-e163f4064c10" xlink:to="loc_us-gaap_FairValueInputsLevel2Member_01a0df8f-19d2-7ce7-b9ca-045f67a7215c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueInputsLevel3Member_01a0df8f-19d2-74ab-b5bd-b63e7104d14f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueInputsLevel3Member"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementsFairValueHierarchyDomain_01a0df8f-19d2-79cf-97c3-e163f4064c10" xlink:to="loc_us-gaap_FairValueInputsLevel3Member_01a0df8f-19d2-74ab-b5bd-b63e7104d14f" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-7af6-91b6-b4cacfec6341" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-7405-9c2c-6891956b9e4c" xlink:to="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-7af6-91b6-b4cacfec6341" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7af6-91b6-b4cacfec6341_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-7af6-91b6-b4cacfec6341" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7af6-91b6-b4cacfec6341_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7b0b-9b54-277394160b8e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-7af6-91b6-b4cacfec6341" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7b0b-9b54-277394160b8e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasurySecuritiesMember_01a0df8f-19d2-71d8-a377-a4eff9280e8a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USTreasurySecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7b0b-9b54-277394160b8e" xlink:to="loc_us-gaap_USTreasurySecuritiesMember_01a0df8f-19d2-71d8-a377-a4eff9280e8a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsAxis_01a0df8f-19d2-7452-a664-27726b41fbc9" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-7405-9c2c-6891956b9e4c" xlink:to="loc_us-gaap_CashAndCashEquivalentsAxis_01a0df8f-19d2-7452-a664-27726b41fbc9" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_01a0df8f-19d2-7452-a664-27726b41fbc9_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_CashAndCashEquivalentsAxis_01a0df8f-19d2-7452-a664-27726b41fbc9" xlink:to="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_01a0df8f-19d2-7452-a664-27726b41fbc9_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_01a0df8f-19d2-7ade-8dc6-4fce2291b250" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_CashAndCashEquivalentsAxis_01a0df8f-19d2-7452-a664-27726b41fbc9" xlink:to="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_01a0df8f-19d2-7ade-8dc6-4fce2291b250" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_CashAndMoneyMarketFundsMember_01a0df8f-19d2-7d31-94c4-22425037fd18" xlink:href="vtgn-20260930.xsd#vtgn_CashAndMoneyMarketFundsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_01a0df8f-19d2-7ade-8dc6-4fce2291b250" xlink:to="loc_vtgn_CashAndMoneyMarketFundsMember_01a0df8f-19d2-7d31-94c4-22425037fd18" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueByMeasurementFrequencyAxis_01a1b2ac-a9b9-76bb-9821-fcd03a6c1907" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueByMeasurementFrequencyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-7405-9c2c-6891956b9e4c" xlink:to="loc_us-gaap_FairValueByMeasurementFrequencyAxis_01a1b2ac-a9b9-76bb-9821-fcd03a6c1907" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementFrequencyDomain_01a1b2ac-a9b9-76bb-9821-fcd03a6c1907_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementFrequencyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FairValueByMeasurementFrequencyAxis_01a1b2ac-a9b9-76bb-9821-fcd03a6c1907" xlink:to="loc_us-gaap_FairValueMeasurementFrequencyDomain_01a1b2ac-a9b9-76bb-9821-fcd03a6c1907_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementFrequencyDomain_01a1b2ac-a9b9-7fbf-9583-41f5f3aa454a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementFrequencyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FairValueByMeasurementFrequencyAxis_01a1b2ac-a9b9-76bb-9821-fcd03a6c1907" xlink:to="loc_us-gaap_FairValueMeasurementFrequencyDomain_01a1b2ac-a9b9-7fbf-9583-41f5f3aa454a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueMeasurementsRecurringMember_01a1b2ad-271f-7856-8eb4-559f7e193f78" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueMeasurementsRecurringMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueMeasurementFrequencyDomain_01a1b2ac-a9b9-7fbf-9583-41f5f3aa454a" xlink:to="loc_us-gaap_FairValueMeasurementsRecurringMember_01a1b2ad-271f-7856-8eb4-559f7e193f78" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CashAndCashEquivalentsFairValueDisclosure_01a0df8f-19d2-740c-b72d-77a177a88be7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CashAndCashEquivalentsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bde-8809-9f199369c09d" xlink:to="loc_us-gaap_CashAndCashEquivalentsFairValueDisclosure_01a0df8f-19d2-740c-b72d-77a177a88be7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_01a0df8f-19d2-7a12-a435-a2f64545186b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bde-8809-9f199369c09d" xlink:to="loc_us-gaap_AvailableForSaleSecuritiesDebtSecurities_01a0df8f-19d2-7a12-a435-a2f64545186b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AssetsFairValueDisclosure_01a0df8f-19d2-7fc7-979f-9c72dc88473b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AssetsFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bde-8809-9f199369c09d" xlink:to="loc_us-gaap_AssetsFairValueDisclosure_01a0df8f-19d2-7fc7-979f-9c72dc88473b" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialLiabilitiesFairValueDisclosure_01a1b2ab-9e13-7f1d-a24a-1329e2e4b004" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialLiabilitiesFairValueDisclosure"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bde-8809-9f199369c09d" xlink:to="loc_us-gaap_FinancialLiabilitiesFairValueDisclosure_01a1b2ab-9e13-7f1d-a24a-1329e2e4b004" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/FairValueMeasurementsMarketableSecuritiesDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#FairValueMeasurementsMarketableSecuritiesDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/FairValueMeasurementsMarketableSecuritiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bcf-bb90-f400b180e678" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-79e9-9666-49ff6b64d801" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bcf-bb90-f400b180e678" xlink:to="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-79e9-9666-49ff6b64d801" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-73bb-868f-f4abdadb896d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_FinancialInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisTable_01a0df8f-19d2-79e9-9666-49ff6b64d801" xlink:to="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-73bb-868f-f4abdadb896d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-73bb-868f-f4abdadb896d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-73bb-868f-f4abdadb896d" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-73bb-868f-f4abdadb896d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7512-9560-31274dbcdd46" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_FinancialInstrumentAxis_01a0df8f-19d2-73bb-868f-f4abdadb896d" xlink:to="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7512-9560-31274dbcdd46" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_USTreasuryNotesSecuritiesMember_01a0df8f-19d2-7383-a6ad-f11702c4d492" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_USTreasuryNotesSecuritiesMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_01a0df8f-19d2-7512-9560-31274dbcdd46" xlink:to="loc_us-gaap_USTreasuryNotesSecuritiesMember_01a0df8f-19d2-7383-a6ad-f11702c4d492" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAmortizedCost_01a0df8f-19d2-7a89-80c7-074a039fa188" xlink:href="vtgn-20260930.xsd#vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAmortizedCost"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bcf-bb90-f400b180e678" xlink:to="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAmortizedCost_01a0df8f-19d2-7a89-80c7-074a039fa188" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAccumulatedGrossUnrealizedGain_01a0df8f-19d2-7a01-a3af-5bec103119fb" xlink:href="vtgn-20260930.xsd#vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAccumulatedGrossUnrealizedGain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bcf-bb90-f400b180e678" xlink:to="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAccumulatedGrossUnrealizedGain_01a0df8f-19d2-7a01-a3af-5bec103119fb" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAccumulatedGrossUnrealizedLossBeforeTax_01a0df8f-19d2-79d2-8850-c8c62d7a5066" xlink:href="vtgn-20260930.xsd#vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAccumulatedGrossUnrealizedLossBeforeTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bcf-bb90-f400b180e678" xlink:to="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalentsAccumulatedGrossUnrealizedLossBeforeTax_01a0df8f-19d2-79d2-8850-c8c62d7a5066" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalents_01a0df8f-19d2-748a-ad0e-9a08f3945978" xlink:href="vtgn-20260930.xsd#vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalents"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisLineItems_01a0df8f-19d2-7bcf-bb90-f400b180e678" xlink:to="loc_vtgn_DebtSecuritiesAvailableForSaleAndCashAndCashEquivalents_01a0df8f-19d2-748a-ad0e-9a08f3945978" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/NotesPayableDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#NotesPayableDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/NotesPayableDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShortTermDebtLineItems_01a0df8f-19d2-7191-af6a-3e85c3206dd2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShortTermDebtLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShortTermDebtTable_01a0df8f-19d2-7dea-9190-e70a6e41fb6a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShortTermDebtTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShortTermDebtLineItems_01a0df8f-19d2-7191-af6a-3e85c3206dd2" xlink:to="loc_us-gaap_ScheduleOfShortTermDebtTable_01a0df8f-19d2-7dea-9190-e70a6e41fb6a" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentAxis_01a0df8f-19d2-7f97-9c2e-4240d04e266e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShortTermDebtTable_01a0df8f-19d2-7dea-9190-e70a6e41fb6a" xlink:to="loc_us-gaap_DebtInstrumentAxis_01a0df8f-19d2-7f97-9c2e-4240d04e266e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_01a0df8f-19d2-7f97-9c2e-4240d04e266e_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_DebtInstrumentAxis_01a0df8f-19d2-7f97-9c2e-4240d04e266e" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_01a0df8f-19d2-7f97-9c2e-4240d04e266e_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentNameDomain_01a0df8f-19d2-7ddb-b467-3193d5418320" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_DebtInstrumentAxis_01a0df8f-19d2-7f97-9c2e-4240d04e266e" xlink:to="loc_us-gaap_DebtInstrumentNameDomain_01a0df8f-19d2-7ddb-b467-3193d5418320" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_PromissoryNoteMember_01a0df8f-19d2-7227-9382-443cdcb804eb" xlink:href="vtgn-20260930.xsd#vtgn_PromissoryNoteMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_DebtInstrumentNameDomain_01a0df8f-19d2-7ddb-b467-3193d5418320" xlink:to="loc_vtgn_PromissoryNoteMember_01a0df8f-19d2-7227-9382-443cdcb804eb" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentInterestRateStatedPercentage_01a0df8f-19d2-79ba-888c-78e83534ce21" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShortTermDebtLineItems_01a0df8f-19d2-7191-af6a-3e85c3206dd2" xlink:to="loc_us-gaap_DebtInstrumentInterestRateStatedPercentage_01a0df8f-19d2-79ba-888c-78e83534ce21" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentFaceAmount_01a0df8f-19d2-7d15-960a-c7e4093ecb8e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentFaceAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShortTermDebtLineItems_01a0df8f-19d2-7191-af6a-3e85c3206dd2" xlink:to="loc_us-gaap_DebtInstrumentFaceAmount_01a0df8f-19d2-7d15-960a-c7e4093ecb8e" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_DebtInstrumentPeriodicPayment_01a0df8f-19d2-7b71-a2a3-2af4f9f69ae5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_DebtInstrumentPeriodicPayment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShortTermDebtLineItems_01a0df8f-19d2-7191-af6a-3e85c3206dd2" xlink:to="loc_us-gaap_DebtInstrumentPeriodicPayment_01a0df8f-19d2-7b71-a2a3-2af4f9f69ae5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_NotesPayableCurrent_01a0df8f-19d2-74c4-9b12-a5f19af75e97" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_NotesPayableCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShortTermDebtLineItems_01a0df8f-19d2-7191-af6a-3e85c3206dd2" xlink:to="loc_us-gaap_NotesPayableCurrent_01a0df8f-19d2-74c4-9b12-a5f19af75e97" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/CapitalStockOctober2023PublicOfferingDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#CapitalStockOctober2023PublicOfferingDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/CapitalStockOctober2023PublicOfferingDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CapitalUnitLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-745d-bc3b-02220716a16e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCapitalUnitsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-745d-bc3b-02220716a16e" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-7dea-bee0-585685cd5636" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-745d-bc3b-02220716a16e" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-7dea-bee0-585685cd5636" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-7dea-bee0-585685cd5636_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-7dea-bee0-585685cd5636" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-7dea-bee0-585685cd5636_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-7ed0-8393-90a7e6062bb7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-7dea-bee0-585685cd5636" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-7ed0-8393-90a7e6062bb7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_PublicOfferingMember_01a0df8f-19d2-7e98-afaf-b550f6e83eef" xlink:href="vtgn-20260930.xsd#vtgn_PublicOfferingMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-7ed0-8393-90a7e6062bb7" xlink:to="loc_vtgn_PublicOfferingMember_01a0df8f-19d2-7e98-afaf-b550f6e83eef" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_OpenMarketSaleAgreementMember_01a0df8f-19d2-7d06-b617-761adf6b1aef" xlink:href="vtgn-20260930.xsd#vtgn_OpenMarketSaleAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-7ed0-8393-90a7e6062bb7" xlink:to="loc_vtgn_OpenMarketSaleAgreementMember_01a0df8f-19d2-7d06-b617-761adf6b1aef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightAxis_01a0df8f-19d2-7672-a5fc-54f54fb38174" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-745d-bc3b-02220716a16e" xlink:to="loc_us-gaap_ClassOfWarrantOrRightAxis_01a0df8f-19d2-7672-a5fc-54f54fb38174" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7672-a5fc-54f54fb38174_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_01a0df8f-19d2-7672-a5fc-54f54fb38174" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7672-a5fc-54f54fb38174_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7334-ba09-e8795f62a1d4" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_01a0df8f-19d2-7672-a5fc-54f54fb38174" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7334-ba09-e8795f62a1d4" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_PrefundedWarrantsMember_01a0df8f-19d2-7dad-845e-10f09ffda0e8" xlink:href="vtgn-20260930.xsd#vtgn_PrefundedWarrantsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7334-ba09-e8795f62a1d4" xlink:to="loc_vtgn_PrefundedWarrantsMember_01a0df8f-19d2-7dad-845e-10f09ffda0e8" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_T1WarrantsMember_01a0df8f-19d2-7d9f-ba44-8fd12b24c29c" xlink:href="vtgn-20260930.xsd#vtgn_T1WarrantsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7334-ba09-e8795f62a1d4" xlink:to="loc_vtgn_T1WarrantsMember_01a0df8f-19d2-7d9f-ba44-8fd12b24c29c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_T2WarrantsMember_01a0df8f-19d2-74b4-b1dc-1378450d9edf" xlink:href="vtgn-20260930.xsd#vtgn_T2WarrantsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_01a0df8f-19d2-7334-ba09-e8795f62a1d4" xlink:to="loc_vtgn_T2WarrantsMember_01a0df8f-19d2-74b4-b1dc-1378450d9edf" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7d6f-aefb-56b462e89871" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-745d-bc3b-02220716a16e" xlink:to="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7d6f-aefb-56b462e89871" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7d6f-aefb-56b462e89871_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7d6f-aefb-56b462e89871" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7d6f-aefb-56b462e89871_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7582-b95b-6b351b10f43f" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7d6f-aefb-56b462e89871" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7582-b95b-6b351b10f43f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_JefferiesLLCMember_01a0df8f-19d2-7661-bfe9-f344d01541e1" xlink:href="vtgn-20260930.xsd#vtgn_JefferiesLLCMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7582-b95b-6b351b10f43f" xlink:to="loc_vtgn_JefferiesLLCMember_01a0df8f-19d2-7661-bfe9-f344d01541e1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_StockSalesAgreementRemainingAmountAvailable_01a0df8f-19d2-776d-8586-3ab46afe01c7" xlink:href="vtgn-20260930.xsd#vtgn_StockSalesAgreementRemainingAmountAvailable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_vtgn_StockSalesAgreementRemainingAmountAvailable_01a0df8f-19d2-776d-8586-3ab46afe01c7" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a0df8f-19d2-7a5f-9f0a-638c323bf7a5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a0df8f-19d2-7a5f-9f0a-638c323bf7a5" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightOutstanding_01a0df8f-19d2-7a7c-b97e-8578dc2558f5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightOutstanding"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_us-gaap_ClassOfWarrantOrRightOutstanding_01a0df8f-19d2-7a7c-b97e-8578dc2558f5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_01a0df8f-19d2-7556-9c41-096a3605a045" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights_01a0df8f-19d2-7556-9c41-096a3605a045" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_01a0df8f-19d2-769c-9fb7-6c4effdbd5b0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1_01a0df8f-19d2-769c-9fb7-6c4effdbd5b0" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_01a0df8f-19d2-71ee-8eee-4fac3bd89ec1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockConsiderationReceivedOnTransaction"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_us-gaap_SaleOfStockConsiderationReceivedOnTransaction_01a0df8f-19d2-71ee-8eee-4fac3bd89ec1" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_WarrantsExercisablePercentageOfCommonSharesOutstandingMinimumBenchmark_01a0df8f-19d2-72f7-b298-2685d96dd125" xlink:href="vtgn-20260930.xsd#vtgn_WarrantsExercisablePercentageOfCommonSharesOutstandingMinimumBenchmark"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_vtgn_WarrantsExercisablePercentageOfCommonSharesOutstandingMinimumBenchmark_01a0df8f-19d2-72f7-b298-2685d96dd125" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_WarrantsExercisablePercentageOfCommonSharesOutstandingMaximumBenchmark_01a0df8f-19d2-7747-bb00-3b93b3a27b3a" xlink:href="vtgn-20260930.xsd#vtgn_WarrantsExercisablePercentageOfCommonSharesOutstandingMaximumBenchmark"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_vtgn_WarrantsExercisablePercentageOfCommonSharesOutstandingMaximumBenchmark_01a0df8f-19d2-7747-bb00-3b93b3a27b3a" xlink:type="arc" order="7"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_WarrantPercentageIncreaseOrDecreaseNoticePeriod_01a0df8f-19d2-7666-a560-c207328207cf" xlink:href="vtgn-20260930.xsd#vtgn_WarrantPercentageIncreaseOrDecreaseNoticePeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7660-ac09-908e4ff2f0da" xlink:to="loc_vtgn_WarrantPercentageIncreaseOrDecreaseNoticePeriod_01a0df8f-19d2-7666-a560-c207328207cf" xlink:type="arc" order="8"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/CapitalStockOpenMarketSaleAgreementDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#CapitalStockOpenMarketSaleAgreementDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/CapitalStockOpenMarketSaleAgreementDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CapitalUnitLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-7c94-9a5b-b142d582b0ee" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCapitalUnitsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:to="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-7c94-9a5b-b142d582b0ee" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7416-9505-ca60d01f5b77" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-7c94-9a5b-b142d582b0ee" xlink:to="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7416-9505-ca60d01f5b77" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7416-9505-ca60d01f5b77_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7416-9505-ca60d01f5b77" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7416-9505-ca60d01f5b77_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-72d8-a8a8-cee071b8d38f" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7416-9505-ca60d01f5b77" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-72d8-a8a8-cee071b8d38f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_JefferiesLLCMember_01a0df8f-19d2-713b-ba5b-93f180567c6b" xlink:href="vtgn-20260930.xsd#vtgn_JefferiesLLCMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-72d8-a8a8-cee071b8d38f" xlink:to="loc_vtgn_JefferiesLLCMember_01a0df8f-19d2-713b-ba5b-93f180567c6b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-78f5-a061-d65267a9afb1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCapitalUnitsTable_01a0df8f-19d2-7c94-9a5b-b142d582b0ee" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-78f5-a061-d65267a9afb1" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-78f5-a061-d65267a9afb1_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-78f5-a061-d65267a9afb1" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-78f5-a061-d65267a9afb1_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-77c8-bb80-e717535012b7" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a0df8f-19d2-78f5-a061-d65267a9afb1" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-77c8-bb80-e717535012b7" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_OpenMarketSaleAgreementMember_01a0df8f-19d2-79f5-b49a-7201e9a15049" xlink:href="vtgn-20260930.xsd#vtgn_OpenMarketSaleAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a0df8f-19d2-77c8-bb80-e717535012b7" xlink:to="loc_vtgn_OpenMarketSaleAgreementMember_01a0df8f-19d2-79f5-b49a-7201e9a15049" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_StockSalesAgreementMaximumAggregateOfferingPrice_01a0df8f-19d2-7ef9-953b-8224f35a463c" xlink:href="vtgn-20260930.xsd#vtgn_StockSalesAgreementMaximumAggregateOfferingPrice"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:to="loc_vtgn_StockSalesAgreementMaximumAggregateOfferingPrice_01a0df8f-19d2-7ef9-953b-8224f35a463c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_StockSalesAgreementRemainingAmountAvailable_01a0df8f-19d2-7580-924a-228e6e16e4af" xlink:href="vtgn-20260930.xsd#vtgn_StockSalesAgreementRemainingAmountAvailable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:to="loc_vtgn_StockSalesAgreementRemainingAmountAvailable_01a0df8f-19d2-7580-924a-228e6e16e4af" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a0df8f-19d2-777e-bfdc-2be31f02c0b0" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a0df8f-19d2-777e-bfdc-2be31f02c0b0" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_01a0df8f-19d2-7499-87f4-022c6341f7ee" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:to="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_01a0df8f-19d2-7499-87f4-022c6341f7ee" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_StockSalesAgreementCommissionOnSharesSoldPercentage_01a0df8f-19d2-7ccf-a319-ca0c4446c28c" xlink:href="vtgn-20260930.xsd#vtgn_StockSalesAgreementCommissionOnSharesSoldPercentage"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CapitalUnitLineItems_01a0df8f-19d2-7cc5-9431-12ccb9a5c0d8" xlink:to="loc_vtgn_StockSalesAgreementCommissionOnSharesSoldPercentage_01a0df8f-19d2-7ccf-a319-ca0c4446c28c" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/CapitalStockNarrativeDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#CapitalStockNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/CapitalStockNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightLineItems_01a1b26c-608b-72d6-ad79-32ecd856c9ba" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightTable_01a1b26c-608b-7002-8759-7fb23ae73276" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_01a1b26c-608b-72d6-ad79-32ecd856c9ba" xlink:to="loc_us-gaap_ClassOfWarrantOrRightTable_01a1b26c-608b-7002-8759-7fb23ae73276" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightAxis_01a1b26c-608b-7fff-99c6-61f304603501" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ClassOfWarrantOrRightTable_01a1b26c-608b-7002-8759-7fb23ae73276" xlink:to="loc_us-gaap_ClassOfWarrantOrRightAxis_01a1b26c-608b-7fff-99c6-61f304603501" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_01a1b26c-608b-7fff-99c6-61f304603501_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_01a1b26c-608b-7fff-99c6-61f304603501" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_01a1b26c-608b-7fff-99c6-61f304603501_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ClassOfWarrantOrRightDomain_01a1b26c-608b-7910-8548-bd164ba0ffce" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ClassOfWarrantOrRightDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_ClassOfWarrantOrRightAxis_01a1b26c-608b-7fff-99c6-61f304603501" xlink:to="loc_us-gaap_ClassOfWarrantOrRightDomain_01a1b26c-608b-7910-8548-bd164ba0ffce" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_T1WarrantsMember_01a1b26d-d2f0-73c4-821b-248bb0db3cf4" xlink:href="vtgn-20260930.xsd#vtgn_T1WarrantsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightDomain_01a1b26c-608b-7910-8548-bd164ba0ffce" xlink:to="loc_vtgn_T1WarrantsMember_01a1b26d-d2f0-73c4-821b-248bb0db3cf4" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ClassOfWarrantOrRightOutstandingWarrantsSubjectToAntiDilutionProtectionFeature_01a1c116-8c6b-7a72-a6ff-82925e8e064e" xlink:href="vtgn-20260930.xsd#vtgn_ClassOfWarrantOrRightOutstandingWarrantsSubjectToAntiDilutionProtectionFeature"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_01a1b26c-608b-72d6-ad79-32ecd856c9ba" xlink:to="loc_vtgn_ClassOfWarrantOrRightOutstandingWarrantsSubjectToAntiDilutionProtectionFeature_01a1c116-8c6b-7a72-a6ff-82925e8e064e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ClassOfWarrantOrRightExpiredInPeriod_01a1b26c-608b-73c5-9645-be074a4f1852" xlink:href="vtgn-20260930.xsd#vtgn_ClassOfWarrantOrRightExpiredInPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_01a1b26c-608b-72d6-ad79-32ecd856c9ba" xlink:to="loc_vtgn_ClassOfWarrantOrRightExpiredInPeriod_01a1b26c-608b-73c5-9645-be074a4f1852" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRightsExpiredDuringPeriod_01a1b26c-bdd8-78fd-a252-da987288402a" xlink:href="vtgn-20260930.xsd#vtgn_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRightsExpiredDuringPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_01a1b26c-608b-72d6-ad79-32ecd856c9ba" xlink:to="loc_vtgn_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRightsExpiredDuringPeriod_01a1b26c-bdd8-78fd-a252-da987288402a" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ClassOfWarrantOrRightExpirationPeriod_01a1b26d-6916-7cc8-b492-5d654e42e295" xlink:href="vtgn-20260930.xsd#vtgn_ClassOfWarrantOrRightExpirationPeriod"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ClassOfWarrantOrRightLineItems_01a1b26c-608b-72d6-ad79-32ecd856c9ba" xlink:to="loc_vtgn_ClassOfWarrantOrRightExpirationPeriod_01a1b26d-6916-7cc8-b492-5d654e42e295" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/StockBasedCompensationScheduleofFairValueofOptionsGrantedDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#StockBasedCompensationScheduleofFairValueofOptionsGrantedDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/StockBasedCompensationScheduleofFairValueofOptionsGrantedDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-7c38-a9a2-74e7071c96ea" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-74c5-a68f-2d3bd41c0e75" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-7c38-a9a2-74e7071c96ea" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-74c5-a68f-2d3bd41c0e75" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-74fa-8a1c-314a2fec0a05" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-74c5-a68f-2d3bd41c0e75" xlink:to="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-74fa-8a1c-314a2fec0a05" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-74fa-8a1c-314a2fec0a05_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-74fa-8a1c-314a2fec0a05" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-74fa-8a1c-314a2fec0a05_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-734b-b55a-ceeb28baa5ae" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-74fa-8a1c-314a2fec0a05" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-734b-b55a-ceeb28baa5ae" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_01a0df8f-19d2-71df-a7ee-09e3d53bf980" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-734b-b55a-ceeb28baa5ae" xlink:to="loc_us-gaap_EmployeeStockOptionMember_01a0df8f-19d2-71df-a7ee-09e3d53bf980" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeAxis_01a0df8f-19d2-7c95-abba-ac989478f2ea" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-74c5-a68f-2d3bd41c0e75" xlink:to="loc_srt_RangeAxis_01a0df8f-19d2-7c95-abba-ac989478f2ea" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_01a0df8f-19d2-7c95-abba-ac989478f2ea_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_RangeAxis_01a0df8f-19d2-7c95-abba-ac989478f2ea" xlink:to="loc_srt_RangeMember_01a0df8f-19d2-7c95-abba-ac989478f2ea_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RangeMember_01a0df8f-19d2-7833-96ed-00103d85ba04" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RangeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_RangeAxis_01a0df8f-19d2-7c95-abba-ac989478f2ea" xlink:to="loc_srt_RangeMember_01a0df8f-19d2-7833-96ed-00103d85ba04" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MinimumMember_01a0df8f-19d2-755c-bf3c-d0a0c606c74b" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MinimumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_01a0df8f-19d2-7833-96ed-00103d85ba04" xlink:to="loc_srt_MinimumMember_01a0df8f-19d2-755c-bf3c-d0a0c606c74b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_MaximumMember_01a0df8f-19d2-74e7-b582-6898d03d0a9c" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_MaximumMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RangeMember_01a0df8f-19d2-7833-96ed-00103d85ba04" xlink:to="loc_srt_MaximumMember_01a0df8f-19d2-74e7-b582-6898d03d0a9c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_01a0df8f-19d2-796e-8b18-8d1b76759556" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-7c38-a9a2-74e7071c96ea" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate_01a0df8f-19d2-796e-8b18-8d1b76759556" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_01a0df8f-19d2-74ed-91df-bf6bf503645c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-7c38-a9a2-74e7071c96ea" xlink:to="loc_us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1_01a0df8f-19d2-74ed-91df-bf6bf503645c" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageVolatilityRate_01a0df8f-19d2-7724-b9f6-edb2dae24fed" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageVolatilityRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-7c38-a9a2-74e7071c96ea" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsWeightedAverageVolatilityRate_01a0df8f-19d2-7724-b9f6-edb2dae24fed" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_01a0df8f-19d2-72c6-98fa-db8f3dd42e0b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-7c38-a9a2-74e7071c96ea" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate_01a0df8f-19d2-72c6-98fa-db8f3dd42e0b" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/StockBasedCompensationScheduleofSummarizesStockBasedCompensationExpenseDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#StockBasedCompensationScheduleofSummarizesStockBasedCompensationExpenseDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/StockBasedCompensationScheduleofSummarizesStockBasedCompensationExpenseDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_01a0df8f-19d2-7cf6-b47a-1ca07bbb54d3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_01a0df8f-19d2-76e8-ac06-b240f551faca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_01a0df8f-19d2-7cf6-b47a-1ca07bbb54d3" xlink:to="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_01a0df8f-19d2-76e8-ac06-b240f551faca" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StatementOfIncomeLocationBalanceAxis_01a0df8f-19d2-7269-ac65-5ef65f1cb761" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StatementOfIncomeLocationBalanceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_01a0df8f-19d2-76e8-ac06-b240f551faca" xlink:to="loc_us-gaap_StatementOfIncomeLocationBalanceAxis_01a0df8f-19d2-7269-ac65-5ef65f1cb761" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_01a0df8f-19d2-7625-a1c5-5e414ddde41d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_01a0df8f-19d2-7cf6-b47a-1ca07bbb54d3" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_01a0df8f-19d2-7625-a1c5-5e414ddde41d" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/StockBasedCompensationScheduleofSummarizesStockBasedCompensationExpenseDetails_1" xlink:type="simple" xlink:href="vtgn-20260930.xsd#StockBasedCompensationScheduleofSummarizesStockBasedCompensationExpenseDetails_1"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/StockBasedCompensationScheduleofSummarizesStockBasedCompensationExpenseDetails_1" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_01a0df8f-19d2-7cf6-b47a-1ca07bbb54d3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_01a0df8f-19d2-76e8-ac06-b240f551faca" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_01a0df8f-19d2-7cf6-b47a-1ca07bbb54d3" xlink:to="loc_us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable_01a0df8f-19d2-76e8-ac06-b240f551faca" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AllocatedShareBasedCompensationExpense_01a0df8f-19d2-7625-a1c5-5e414ddde41d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AllocatedShareBasedCompensationExpense"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems_01a0df8f-19d2-7cf6-b47a-1ca07bbb54d3" xlink:to="loc_us-gaap_AllocatedShareBasedCompensationExpense_01a0df8f-19d2-7625-a1c5-5e414ddde41d" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/StockBasedCompensationNarrativeDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#StockBasedCompensationNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/StockBasedCompensationNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-7ca1-8c5e-d0d5f1cdbb51" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:to="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-7ca1-8c5e-d0d5f1cdbb51" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-7b65-8b8a-9a017a180587" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AwardTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-7ca1-8c5e-d0d5f1cdbb51" xlink:to="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-7b65-8b8a-9a017a180587" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-7b65-8b8a-9a017a180587_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-7b65-8b8a-9a017a180587" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-7b65-8b8a-9a017a180587_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-72d2-85be-62f1eea07d38" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AwardTypeAxis_01a0df8f-19d2-7b65-8b8a-9a017a180587" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-72d2-85be-62f1eea07d38" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_01a0df8f-19d2-749e-8e68-a07694fbd9e1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_01a0df8f-19d2-72d2-85be-62f1eea07d38" xlink:to="loc_us-gaap_EmployeeStockOptionMember_01a0df8f-19d2-749e-8e68-a07694fbd9e1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GranteeStatusAxis_01a0df8f-19d2-71a2-aafd-810374a33cc5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable_01a0df8f-19d2-7ca1-8c5e-d0d5f1cdbb51" xlink:to="loc_us-gaap_GranteeStatusAxis_01a0df8f-19d2-71a2-aafd-810374a33cc5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GranteeStatusDomain_01a0df8f-19d2-71a2-aafd-810374a33cc5_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_GranteeStatusAxis_01a0df8f-19d2-71a2-aafd-810374a33cc5" xlink:to="loc_us-gaap_GranteeStatusDomain_01a0df8f-19d2-71a2-aafd-810374a33cc5_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_GranteeStatusDomain_01a0df8f-19d2-76db-bde8-b99b0f9e4d09" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_GranteeStatusDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_GranteeStatusAxis_01a0df8f-19d2-71a2-aafd-810374a33cc5" xlink:to="loc_us-gaap_GranteeStatusDomain_01a0df8f-19d2-76db-bde8-b99b0f9e4d09" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedPaymentArrangementEmployeeMember_01a0df8f-19d2-7c85-a339-52c164079946" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedPaymentArrangementEmployeeMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_GranteeStatusDomain_01a0df8f-19d2-76db-bde8-b99b0f9e4d09" xlink:to="loc_us-gaap_ShareBasedPaymentArrangementEmployeeMember_01a0df8f-19d2-7c85-a339-52c164079946" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_01a0df8f-19d2-76eb-8f4e-4e1d3582ff87" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue_01a0df8f-19d2-76eb-8f4e-4e1d3582ff87" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_01a0df8f-19d2-76f6-94c0-24c39fc86004" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised_01a0df8f-19d2-76f6-94c0-24c39fc86004" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_01a0df8f-19d2-7ead-9353-dab10fee9446" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions_01a0df8f-19d2-7ead-9353-dab10fee9446" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_01a0df8f-19d2-720b-97ce-0d8d75de975e" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:to="loc_us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1_01a0df8f-19d2-720b-97ce-0d8d75de975e" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_01a0df8f-19d2-716f-a985-1bbcf290a1ec" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems_01a0df8f-19d2-79f6-b535-fe6c7527221e" xlink:to="loc_us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodGross_01a0df8f-19d2-716f-a985-1bbcf290a1ec" xlink:type="arc" order="4"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/NetLossperCommonShareScheduleofAntidilutiveSecuritiesExcludedfromComputationofEarningsPerShareDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#NetLossperCommonShareScheduleofAntidilutiveSecuritiesExcludedfromComputationofEarningsPerShareDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/NetLossperCommonShareScheduleofAntidilutiveSecuritiesExcludedfromComputationofEarningsPerShareDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_01a0df8f-19d2-73f0-926a-9cf98109813b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01a0df8f-19d2-78fd-b961-60dcacc5a88f" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_01a0df8f-19d2-73f0-926a-9cf98109813b" xlink:to="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01a0df8f-19d2-78fd-b961-60dcacc5a88f" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_01a0df8f-19d2-790d-acae-6e31b2c931af" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01a0df8f-19d2-78fd-b961-60dcacc5a88f" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_01a0df8f-19d2-790d-acae-6e31b2c931af" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_01a0df8f-19d2-790d-acae-6e31b2c931af_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_01a0df8f-19d2-790d-acae-6e31b2c931af" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_01a0df8f-19d2-790d-acae-6e31b2c931af_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesNameDomain_01a0df8f-19d2-7422-93f9-5aefb5f471f3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis_01a0df8f-19d2-790d-acae-6e31b2c931af" xlink:to="loc_us-gaap_AntidilutiveSecuritiesNameDomain_01a0df8f-19d2-7422-93f9-5aefb5f471f3" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeStockOptionMember_01a0df8f-19d2-71a9-9eba-f9faf6d8b1a3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeStockOptionMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_01a0df8f-19d2-7422-93f9-5aefb5f471f3" xlink:to="loc_us-gaap_EmployeeStockOptionMember_01a0df8f-19d2-71a9-9eba-f9faf6d8b1a3" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_WarrantMember_01a0df8f-19d2-7499-9b54-5d620088c116" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_WarrantMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesNameDomain_01a0df8f-19d2-7422-93f9-5aefb5f471f3" xlink:to="loc_us-gaap_WarrantMember_01a0df8f-19d2-7499-9b54-5d620088c116" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameAxis_01a0df8f-19d2-7674-9ece-c69925228b74" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable_01a0df8f-19d2-78fd-b961-60dcacc5a88f" xlink:to="loc_us-gaap_PlanNameAxis_01a0df8f-19d2-7674-9ece-c69925228b74" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_01a0df8f-19d2-7674-9ece-c69925228b74_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_PlanNameAxis_01a0df8f-19d2-7674-9ece-c69925228b74" xlink:to="loc_us-gaap_PlanNameDomain_01a0df8f-19d2-7674-9ece-c69925228b74_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_PlanNameDomain_01a0df8f-19d2-7d09-b458-b6496d86e392" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_PlanNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_PlanNameAxis_01a0df8f-19d2-7674-9ece-c69925228b74" xlink:to="loc_us-gaap_PlanNameDomain_01a0df8f-19d2-7d09-b458-b6496d86e392" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_AmendedAndRestated2016StockIncentivePlanAndAmendedAndRestated2019OmnibusEquityIncentivePlanMember_01a0df8f-19d2-7d52-82a7-6e73a1fc37fa" xlink:href="vtgn-20260930.xsd#vtgn_AmendedAndRestated2016StockIncentivePlanAndAmendedAndRestated2019OmnibusEquityIncentivePlanMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_PlanNameDomain_01a0df8f-19d2-7d09-b458-b6496d86e392" xlink:to="loc_vtgn_AmendedAndRestated2016StockIncentivePlanAndAmendedAndRestated2019OmnibusEquityIncentivePlanMember_01a0df8f-19d2-7d52-82a7-6e73a1fc37fa" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_01a0df8f-19d2-7160-b513-6b7fc065cd7b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems_01a0df8f-19d2-73f0-926a-9cf98109813b" xlink:to="loc_us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount_01a0df8f-19d2-7160-b513-6b7fc065cd7b" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/SublicensingandCollaborativeAgreementsScheduleofChangesinContractAssetsandContractLiabilitiesDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#SublicensingandCollaborativeAgreementsScheduleofChangesinContractAssetsandContractLiabilitiesDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/SublicensingandCollaborativeAgreementsScheduleofChangesinContractAssetsandContractLiabilitiesDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7702-993f-49376eee5089" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-7e58-9ae1-45567240e0c3" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7702-993f-49376eee5089" xlink:to="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-7e58-9ae1-45567240e0c3" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-709a-a1a2-aa5e3db0905d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-7e58-9ae1-45567240e0c3" xlink:to="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-709a-a1a2-aa5e3db0905d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-709a-a1a2-aa5e3db0905d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-709a-a1a2-aa5e3db0905d" xlink:to="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-709a-a1a2-aa5e3db0905d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-7eec-8645-38a3cb576df2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-709a-a1a2-aa5e3db0905d" xlink:to="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-7eec-8645-38a3cb576df2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CollaborativeArrangementMember_01a0df8f-19d2-745e-9f4f-0bb81414c1d1" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-7eec-8645-38a3cb576df2" xlink:to="loc_us-gaap_CollaborativeArrangementMember_01a0df8f-19d2-745e-9f4f-0bb81414c1d1" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ContractWithCustomerAssetRollForward_01a0df8f-19d2-773c-b83e-699762c3626d" xlink:href="vtgn-20260930.xsd#vtgn_ContractWithCustomerAssetRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7702-993f-49376eee5089" xlink:to="loc_vtgn_ContractWithCustomerAssetRollForward_01a0df8f-19d2-773c-b83e-699762c3626d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerAssetNet_01a0df8f-19d2-7ab6-bcb7-a9bbe9f59358" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractWithCustomerAssetNet"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_vtgn_ContractWithCustomerAssetRollForward_01a0df8f-19d2-773c-b83e-699762c3626d" xlink:to="loc_us-gaap_ContractWithCustomerAssetNet_01a0df8f-19d2-7ab6-bcb7-a9bbe9f59358" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ContractWithCustomerAssetIncreaseForAdditions_01a0df8f-19d2-706c-8f8b-8784173ab532" xlink:href="vtgn-20260930.xsd#vtgn_ContractWithCustomerAssetIncreaseForAdditions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_vtgn_ContractWithCustomerAssetRollForward_01a0df8f-19d2-773c-b83e-699762c3626d" xlink:to="loc_vtgn_ContractWithCustomerAssetIncreaseForAdditions_01a0df8f-19d2-706c-8f8b-8784173ab532" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ContractWithCustomerAssetDecreaseForDeductions_01a0df8f-19d2-7fd3-a8e2-dc0f096dd789" xlink:href="vtgn-20260930.xsd#vtgn_ContractWithCustomerAssetDecreaseForDeductions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_vtgn_ContractWithCustomerAssetRollForward_01a0df8f-19d2-773c-b83e-699762c3626d" xlink:to="loc_vtgn_ContractWithCustomerAssetDecreaseForDeductions_01a0df8f-19d2-7fd3-a8e2-dc0f096dd789" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerAssetNet_01a0df8f-19d2-700e-8623-48a094b0e763" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractWithCustomerAssetNet"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ContractWithCustomerLiabilitiesRollForward_01a0df8f-19d2-7a7c-9868-56883a127e97" xlink:href="vtgn-20260930.xsd#vtgn_ContractWithCustomerLiabilitiesRollForward"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7702-993f-49376eee5089" xlink:to="loc_vtgn_ContractWithCustomerLiabilitiesRollForward_01a0df8f-19d2-7a7c-9868-56883a127e97" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerLiability_01a0df8f-19d2-7f46-881d-ea390f7f61b2" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractWithCustomerLiability"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_vtgn_ContractWithCustomerLiabilitiesRollForward_01a0df8f-19d2-7a7c-9868-56883a127e97" xlink:to="loc_us-gaap_ContractWithCustomerLiability_01a0df8f-19d2-7f46-881d-ea390f7f61b2" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ContractWithCustomerLiabilityIncreaseForAdditions_01a0df8f-19d2-7db4-8a0d-fb70b50fa6a5" xlink:href="vtgn-20260930.xsd#vtgn_ContractWithCustomerLiabilityIncreaseForAdditions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_vtgn_ContractWithCustomerLiabilitiesRollForward_01a0df8f-19d2-7a7c-9868-56883a127e97" xlink:to="loc_vtgn_ContractWithCustomerLiabilityIncreaseForAdditions_01a0df8f-19d2-7db4-8a0d-fb70b50fa6a5" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_ContractWithCustomerLiabilityDecreaseForDeductions_01a0df8f-19d2-73ba-8d02-15ba9dc1abf1" xlink:href="vtgn-20260930.xsd#vtgn_ContractWithCustomerLiabilityDecreaseForDeductions"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_vtgn_ContractWithCustomerLiabilitiesRollForward_01a0df8f-19d2-7a7c-9868-56883a127e97" xlink:to="loc_vtgn_ContractWithCustomerLiabilityDecreaseForDeductions_01a0df8f-19d2-73ba-8d02-15ba9dc1abf1" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerLiability_01a0df8f-19d2-78aa-9633-81bfd789f006" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractWithCustomerLiability"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/SublicensingandCollaborativeAgreementsNarrativeDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#SublicensingandCollaborativeAgreementsNarrativeDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/SublicensingandCollaborativeAgreementsNarrativeDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-737b-aaee-db252c6e85b5" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-737b-aaee-db252c6e85b5" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductOrServiceAxis_01a0df8f-19d2-70aa-8e1b-0b945452dbad" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductOrServiceAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-737b-aaee-db252c6e85b5" xlink:to="loc_srt_ProductOrServiceAxis_01a0df8f-19d2-70aa-8e1b-0b945452dbad" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_01a0df8f-19d2-70aa-8e1b-0b945452dbad_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_ProductOrServiceAxis_01a0df8f-19d2-70aa-8e1b-0b945452dbad" xlink:to="loc_srt_ProductsAndServicesDomain_01a0df8f-19d2-70aa-8e1b-0b945452dbad_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain_01a0df8f-19d2-7873-8676-72c3faca8e60" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductsAndServicesDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_ProductOrServiceAxis_01a0df8f-19d2-70aa-8e1b-0b945452dbad" xlink:to="loc_srt_ProductsAndServicesDomain_01a0df8f-19d2-7873-8676-72c3faca8e60" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_LicenseMember_01a0df8f-19d2-7a87-94c5-13c267bae80a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_LicenseMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain_01a0df8f-19d2-7873-8676-72c3faca8e60" xlink:to="loc_us-gaap_LicenseMember_01a0df8f-19d2-7a87-94c5-13c267bae80a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-75f7-a57a-4e0adc7ab01d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfArrangementAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-737b-aaee-db252c6e85b5" xlink:to="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-75f7-a57a-4e0adc7ab01d" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-75f7-a57a-4e0adc7ab01d_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-75f7-a57a-4e0adc7ab01d" xlink:to="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-75f7-a57a-4e0adc7ab01d_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-792b-99ae-fadc5c3f8e7b" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_TypeOfArrangementAxis_01a0df8f-19d2-75f7-a57a-4e0adc7ab01d" xlink:to="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-792b-99ae-fadc5c3f8e7b" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_CollaborativeArrangementAffaMedAgreementMember_01a0df8f-19d2-7f09-9e18-7458c506e6b5" xlink:href="vtgn-20260930.xsd#vtgn_CollaborativeArrangementAffaMedAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-792b-99ae-fadc5c3f8e7b" xlink:to="loc_vtgn_CollaborativeArrangementAffaMedAgreementMember_01a0df8f-19d2-7f09-9e18-7458c506e6b5" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_NegotiationAgreementMember_01a0df8f-19d2-7985-9443-07a5a97f261f" xlink:href="vtgn-20260930.xsd#vtgn_NegotiationAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_ArrangementsAndNonarrangementTransactionsMember_01a0df8f-19d2-792b-99ae-fadc5c3f8e7b" xlink:to="loc_vtgn_NegotiationAgreementMember_01a0df8f-19d2-7985-9443-07a5a97f261f" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7189-8689-06488c1738db" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfCollaborativeArrangementsAndNoncollaborativeArrangementTransactionsTable_01a0df8f-19d2-737b-aaee-db252c6e85b5" xlink:to="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7189-8689-06488c1738db" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7189-8689-06488c1738db_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7189-8689-06488c1738db" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7189-8689-06488c1738db_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7ce5-bc7a-aae69b203b80" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_01a0df8f-19d2-7189-8689-06488c1738db" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7ce5-bc7a-aae69b203b80" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_FujiPharmaMember_01a0df8f-19d2-7e7b-a5a3-17a639cc993d" xlink:href="vtgn-20260930.xsd#vtgn_FujiPharmaMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a0df8f-19d2-7ce5-bc7a-aae69b203b80" xlink:to="loc_vtgn_FujiPharmaMember_01a0df8f-19d2-7e7b-a5a3-17a639cc993d" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_UpfrontLicensePayment_01a0df8f-19d2-7f52-9851-0e49e64b636b" xlink:href="vtgn-20260930.xsd#vtgn_UpfrontLicensePayment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_vtgn_UpfrontLicensePayment_01a0df8f-19d2-7f52-9851-0e49e64b636b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_RevenueRecognitionMilestoneMethodMaximumMilestonePaymentsPermitted_01a0df8f-19d2-7c88-a9da-c7cd9b55ccb0" xlink:href="vtgn-20260930.xsd#vtgn_RevenueRecognitionMilestoneMethodMaximumMilestonePaymentsPermitted"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_vtgn_RevenueRecognitionMilestoneMethodMaximumMilestonePaymentsPermitted_01a0df8f-19d2-7c88-a9da-c7cd9b55ccb0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerLiabilityCurrent_01a0df8f-19d2-7785-8390-8abfb9433c51" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractWithCustomerLiabilityCurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_us-gaap_ContractWithCustomerLiabilityCurrent_01a0df8f-19d2-7785-8390-8abfb9433c51" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ContractWithCustomerLiabilityNoncurrent_01a0df8f-19d2-716f-8420-0b652eb9ff83" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ContractWithCustomerLiabilityNoncurrent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_us-gaap_ContractWithCustomerLiabilityNoncurrent_01a0df8f-19d2-716f-8420-0b652eb9ff83" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax_01a0df8f-19d2-70ba-b025-521fe613f4bc" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_us-gaap_RevenueFromContractWithCustomerIncludingAssessedTax_01a0df8f-19d2-70ba-b025-521fe613f4bc" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_CollaborativeArrangementTerminationPeriodOptionOne_01a0df8f-19d2-7a9e-97de-f102332d4b71" xlink:href="vtgn-20260930.xsd#vtgn_CollaborativeArrangementTerminationPeriodOptionOne"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_vtgn_CollaborativeArrangementTerminationPeriodOptionOne_01a0df8f-19d2-7a9e-97de-f102332d4b71" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_CollaborativeArrangementTerminationPeriodOptionTwo_01a0df8f-19d2-7afd-aa58-33225c846906" xlink:href="vtgn-20260930.xsd#vtgn_CollaborativeArrangementTerminationPeriodOptionTwo"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_vtgn_CollaborativeArrangementTerminationPeriodOptionTwo_01a0df8f-19d2-7afd-aa58-33225c846906" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_CollaborativeArrangementUpfrontPayment_01a0df8f-19d2-79bc-921b-28bc8c38481a" xlink:href="vtgn-20260930.xsd#vtgn_CollaborativeArrangementUpfrontPayment"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_CollaborativeArrangementsAndNoncollaborativeArrangementTransactionsLineItems_01a0df8f-19d2-7a3f-b5ad-db4bce9d774d" xlink:to="loc_vtgn_CollaborativeArrangementUpfrontPayment_01a0df8f-19d2-79bc-921b-28bc8c38481a" xlink:type="arc" order="7"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/RelatedPartyTransactionsDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#RelatedPartyTransactionsDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/RelatedPartyTransactionsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyTransactionLineItems_01a0df8f-19d3-73a5-9b81-a812006087d6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_01a0df8f-19d3-7a4b-bf11-3180b49404fe" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_01a0df8f-19d3-73a5-9b81-a812006087d6" xlink:to="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_01a0df8f-19d3-7a4b-bf11-3180b49404fe" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_01a0df8f-19d3-7236-93d7-52222fd32f42" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_01a0df8f-19d3-7a4b-bf11-3180b49404fe" xlink:to="loc_us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_01a0df8f-19d3-7236-93d7-52222fd32f42" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyDomain_01a0df8f-19d3-7236-93d7-52222fd32f42_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_01a0df8f-19d3-7236-93d7-52222fd32f42" xlink:to="loc_us-gaap_RelatedPartyDomain_01a0df8f-19d3-7236-93d7-52222fd32f42_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyDomain_01a0df8f-19d3-72ad-a2c0-d518a9dcb049" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RelatedPartyTransactionsByRelatedPartyAxis_01a0df8f-19d3-7236-93d7-52222fd32f42" xlink:to="loc_us-gaap_RelatedPartyDomain_01a0df8f-19d3-72ad-a2c0-d518a9dcb049" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyMember_01a0df8f-19d3-7916-b74b-b97a69cbaa8a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyDomain_01a0df8f-19d3-72ad-a2c0-d518a9dcb049" xlink:to="loc_us-gaap_RelatedPartyMember_01a0df8f-19d3-7916-b74b-b97a69cbaa8a" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyTransactionAxis_01a0df8f-19d3-78fe-92e2-231204dcd821" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_ScheduleOfRelatedPartyTransactionsByRelatedPartyTable_01a0df8f-19d3-7a4b-bf11-3180b49404fe" xlink:to="loc_us-gaap_RelatedPartyTransactionAxis_01a0df8f-19d3-78fe-92e2-231204dcd821" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyTransactionDomain_01a0df8f-19d3-78fe-92e2-231204dcd821_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RelatedPartyTransactionAxis_01a0df8f-19d3-78fe-92e2-231204dcd821" xlink:to="loc_us-gaap_RelatedPartyTransactionDomain_01a0df8f-19d3-78fe-92e2-231204dcd821_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RelatedPartyTransactionDomain_01a0df8f-19d3-72ec-8490-7cee1376308d" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RelatedPartyTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RelatedPartyTransactionAxis_01a0df8f-19d3-78fe-92e2-231204dcd821" xlink:to="loc_us-gaap_RelatedPartyTransactionDomain_01a0df8f-19d3-72ec-8490-7cee1376308d" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_DotsonConsultingAgreementMember_01a0df8f-19d3-7b48-bec7-530d1e1bdfad" xlink:href="vtgn-20260930.xsd#vtgn_DotsonConsultingAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionDomain_01a0df8f-19d3-72ec-8490-7cee1376308d" xlink:to="loc_vtgn_DotsonConsultingAgreementMember_01a0df8f-19d3-7b48-bec7-530d1e1bdfad" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_OperatingCostsAndExpenses_01a0df8f-19d3-7be6-8551-83afce4da408" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_OperatingCostsAndExpenses"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RelatedPartyTransactionLineItems_01a0df8f-19d3-73a5-9b81-a812006087d6" xlink:to="loc_us-gaap_OperatingCostsAndExpenses_01a0df8f-19d3-7be6-8551-83afce4da408" xlink:type="arc" order="0"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://www.vistagen.com/role/SubsequentEventsDetails" xlink:type="simple" xlink:href="vtgn-20260930.xsd#SubsequentEventsDetails"/>
  <link:definitionLink xlink:role="http://www.vistagen.com/role/SubsequentEventsDetails" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventLineItems_01a1c10b-57a8-7fd3-b209-fd6cf3a2ba80" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventLineItems"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTable"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="loc_us-gaap_SubsequentEventLineItems_01a1c10b-57a8-7fd3-b209-fd6cf3a2ba80" xlink:to="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:type="arc" order="1" xbrldt:closed="true" xbrldt:contextElement="segment"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestructuringPlanAxis_01a1c10b-57a8-7958-b268-7fe47cef63df" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestructuringPlanAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:to="loc_us-gaap_RestructuringPlanAxis_01a1c10b-57a8-7958-b268-7fe47cef63df" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestructuringPlanDomain_01a1c10b-57a8-7958-b268-7fe47cef63df_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestructuringPlanDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RestructuringPlanAxis_01a1c10b-57a8-7958-b268-7fe47cef63df" xlink:to="loc_us-gaap_RestructuringPlanDomain_01a1c10b-57a8-7958-b268-7fe47cef63df_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestructuringPlanDomain_01a1c10b-57a8-77c3-8cb9-898a76de3cef" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestructuringPlanDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RestructuringPlanAxis_01a1c10b-57a8-7958-b268-7fe47cef63df" xlink:to="loc_us-gaap_RestructuringPlanDomain_01a1c10b-57a8-77c3-8cb9-898a76de3cef" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_October2026RIFMember_01a1c10c-3ab2-74bd-ae1f-bdeca5529c1e" xlink:href="vtgn-20260930.xsd#vtgn_October2026RIFMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_RestructuringPlanDomain_01a1c10b-57a8-77c3-8cb9-898a76de3cef" xlink:to="loc_vtgn_October2026RIFMember_01a1c10c-3ab2-74bd-ae1f-bdeca5529c1e" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestructuringCostAndReserveAxis_01a1c10b-8572-766a-9013-07b22e5cbd18" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestructuringCostAndReserveAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:to="loc_us-gaap_RestructuringCostAndReserveAxis_01a1c10b-8572-766a-9013-07b22e5cbd18" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TypeOfRestructuringDomain_01a1c10b-8572-766a-9013-07b22e5cbd18_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfRestructuringDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_RestructuringCostAndReserveAxis_01a1c10b-8572-766a-9013-07b22e5cbd18" xlink:to="loc_us-gaap_TypeOfRestructuringDomain_01a1c10b-8572-766a-9013-07b22e5cbd18_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_TypeOfRestructuringDomain_01a1c10b-8572-76d1-809e-397594785d0a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_TypeOfRestructuringDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_RestructuringCostAndReserveAxis_01a1c10b-8572-766a-9013-07b22e5cbd18" xlink:to="loc_us-gaap_TypeOfRestructuringDomain_01a1c10b-8572-76d1-809e-397594785d0a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_EmployeeSeveranceMember_01a1c10b-f258-7256-a961-7a4ee5e9a623" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_EmployeeSeveranceMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_TypeOfRestructuringDomain_01a1c10b-8572-76d1-809e-397594785d0a" xlink:to="loc_us-gaap_EmployeeSeveranceMember_01a1c10b-f258-7256-a961-7a4ee5e9a623" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_StatementScenarioAxis_01a1c10c-87b4-7364-b3b5-b036988eeaa3" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_StatementScenarioAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:to="loc_srt_StatementScenarioAxis_01a1c10c-87b4-7364-b3b5-b036988eeaa3" xlink:type="arc" order="3"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ScenarioUnspecifiedDomain_01a1c10c-87b4-7364-b3b5-b036988eeaa3_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioUnspecifiedDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_StatementScenarioAxis_01a1c10c-87b4-7364-b3b5-b036988eeaa3" xlink:to="loc_srt_ScenarioUnspecifiedDomain_01a1c10c-87b4-7364-b3b5-b036988eeaa3_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ScenarioUnspecifiedDomain_01a1c10c-87b4-730b-9cbb-19ea29e99f41" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioUnspecifiedDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_StatementScenarioAxis_01a1c10c-87b4-7364-b3b5-b036988eeaa3" xlink:to="loc_srt_ScenarioUnspecifiedDomain_01a1c10c-87b4-730b-9cbb-19ea29e99f41" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_ScenarioForecastMember_01a1c10c-d7aa-7afd-a6cd-8003e526508b" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ScenarioForecastMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ScenarioUnspecifiedDomain_01a1c10c-87b4-730b-9cbb-19ea29e99f41" xlink:to="loc_srt_ScenarioForecastMember_01a1c10c-d7aa-7afd-a6cd-8003e526508b" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeAxis_01a1c10c-a3d8-70d6-b135-0c44abdb7690" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:to="loc_us-gaap_SubsequentEventTypeAxis_01a1c10c-a3d8-70d6-b135-0c44abdb7690" xlink:type="arc" order="4"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_01a1c10c-a3d8-70d6-b135-0c44abdb7690_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_01a1c10c-a3d8-70d6-b135-0c44abdb7690" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_01a1c10c-a3d8-70d6-b135-0c44abdb7690_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventTypeDomain_01a1c10c-a3d8-7045-9cc4-724eebe37e1a" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventTypeDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsequentEventTypeAxis_01a1c10c-a3d8-70d6-b135-0c44abdb7690" xlink:to="loc_us-gaap_SubsequentEventTypeDomain_01a1c10c-a3d8-7045-9cc4-724eebe37e1a" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsequentEventMember_01a1c10c-c082-7708-a2ce-909aaae66017" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsequentEventMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventTypeDomain_01a1c10c-a3d8-7045-9cc4-724eebe37e1a" xlink:to="loc_us-gaap_SubsequentEventMember_01a1c10c-c082-7708-a2ce-909aaae66017" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_CounterpartyNameAxis_01a1c10e-6fed-70b6-8a92-ab25a9d45ac3" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_CounterpartyNameAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:to="loc_srt_CounterpartyNameAxis_01a1c10e-6fed-70b6-8a92-ab25a9d45ac3" xlink:type="arc" order="5"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a1c10e-6fed-70b6-8a92-ab25a9d45ac3_default" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_srt_CounterpartyNameAxis_01a1c10e-6fed-70b6-8a92-ab25a9d45ac3" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a1c10e-6fed-70b6-8a92-ab25a9d45ac3_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a1c10e-6fed-7529-8f1f-5e6592cad8f0" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_RepurchaseAgreementCounterpartyNameDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_srt_CounterpartyNameAxis_01a1c10e-6fed-70b6-8a92-ab25a9d45ac3" xlink:to="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a1c10e-6fed-7529-8f1f-5e6592cad8f0" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_JefferiesLLCMember_01a1c10e-a6f5-7e50-abfa-4e50ce284971" xlink:href="vtgn-20260930.xsd#vtgn_JefferiesLLCMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_RepurchaseAgreementCounterpartyNameDomain_01a1c10e-6fed-7529-8f1f-5e6592cad8f0" xlink:to="loc_vtgn_JefferiesLLCMember_01a1c10e-a6f5-7e50-abfa-4e50ce284971" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SubsidiarySaleOfStockAxis_01a1c10e-9450-7f95-8145-50ed8b334136" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SubsidiarySaleOfStockAxis"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="loc_us-gaap_SubsequentEventTable_01a1c10b-57a8-79fd-8819-3a04b793e819" xlink:to="loc_us-gaap_SubsidiarySaleOfStockAxis_01a1c10e-9450-7f95-8145-50ed8b334136" xlink:type="arc" order="6"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a1c10e-9450-7f95-8145-50ed8b334136_default" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a1c10e-9450-7f95-8145-50ed8b334136" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a1c10e-9450-7f95-8145-50ed8b334136_default" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a1c10e-9450-7047-85ae-e74b27f589ee" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_SaleOfStockNameOfTransactionDomain"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="loc_us-gaap_SubsidiarySaleOfStockAxis_01a1c10e-9450-7f95-8145-50ed8b334136" xlink:to="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a1c10e-9450-7047-85ae-e74b27f589ee" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_OpenMarketSaleAgreementMember_01a1c10e-bd8b-7224-8c86-780a6694b3de" xlink:href="vtgn-20260930.xsd#vtgn_OpenMarketSaleAgreementMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SaleOfStockNameOfTransactionDomain_01a1c10e-9450-7047-85ae-e74b27f589ee" xlink:to="loc_vtgn_OpenMarketSaleAgreementMember_01a1c10e-bd8b-7224-8c86-780a6694b3de" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent_01a1c10a-e513-7d2b-ac97-26f9fbd88e3c" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_01a1c10b-57a8-7fd3-b209-fd6cf3a2ba80" xlink:to="loc_us-gaap_RestructuringAndRelatedCostNumberOfPositionsEliminatedPeriodPercent_01a1c10a-e513-7d2b-ac97-26f9fbd88e3c" xlink:type="arc" order="0"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_RestructuringAndRelatedCostExpectedCost1_01a1c109-f787-7ab2-92f3-a760352fdd46" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_RestructuringAndRelatedCostExpectedCost1"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_01a1c10b-57a8-7fd3-b209-fd6cf3a2ba80" xlink:to="loc_us-gaap_RestructuringAndRelatedCostExpectedCost1_01a1c109-f787-7ab2-92f3-a760352fdd46" xlink:type="arc" order="1"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a1c10e-4735-7040-b0bc-dc8a13d093b8" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_01a1c10b-57a8-7fd3-b209-fd6cf3a2ba80" xlink:to="loc_us-gaap_StockIssuedDuringPeriodSharesNewIssues_01a1c10e-4735-7040-b0bc-dc8a13d093b8" xlink:type="arc" order="2"/>
    <link:loc xlink:type="locator" xlink:label="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_01a1c10f-4656-7d81-98d4-be5f63de66e6" xlink:href="https://xbrl.fasb.org/us-gaap/2026/elts/us-gaap-2026.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_us-gaap_SubsequentEventLineItems_01a1c10b-57a8-7fd3-b209-fd6cf3a2ba80" xlink:to="loc_us-gaap_ProceedsFromIssuanceOfCommonStock_01a1c10f-4656-7d81-98d4-be5f63de66e6" xlink:type="arc" order="3"/>
  </link:definitionLink>
  <link:roleRef roleURI="http://fasb.org/srt/role/srt-eedm/ExtensibleEnumerationLists" xlink:type="simple" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-roles-2026.xsd#eedm1"/>
  <link:definitionLink xlink:role="http://fasb.org/srt/role/srt-eedm/ExtensibleEnumerationLists" xlink:type="extended">
    <link:loc xlink:type="locator" xlink:label="loc_srt_ProductsAndServicesDomain" xlink:href="https://xbrl.fasb.org/srt/2026/elts/srt-2026.xsd#srt_ProductsAndServicesDomain"/>
    <link:loc xlink:type="locator" xlink:label="loc_vtgn_SublicenseAndOtherRevenueMember_01a0df8f-191b-7f09-a2d0-d1949a60e7e2" xlink:href="vtgn-20260930.xsd#vtgn_SublicenseAndOtherRevenueMember"/>
    <link:definitionArc xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="loc_srt_ProductsAndServicesDomain" xlink:to="loc_vtgn_SublicenseAndOtherRevenueMember_01a0df8f-191b-7f09-a2d0-d1949a60e7e2" xlink:type="arc" order="501"/>
  </link:definitionLink>
</link:linkbase>
