<?xml version="1.0"?>
<ownershipDocument>

    <schemaVersion>X0609</schemaVersion>

    <documentType>4</documentType>

    <periodOfReport>2026-07-01</periodOfReport>

    <notSubjectToSection16>0</notSubjectToSection16>

    <issuer>
        <issuerCik>0001335105</issuerCik>
        <issuerName>LIXTE BIOTECHNOLOGY HOLDINGS, INC.</issuerName>
        <issuerTradingSymbol>LIXT</issuerTradingSymbol>
        <issuerForeignTradingSymbol></issuerForeignTradingSymbol>
    </issuer>

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001569735</rptOwnerCik>
            <rptOwnerName>Felix Lourdes</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerNonUSAddressFlag>false</rptOwnerNonUSAddressFlag>
            <rptOwnerStreet1>433 PLAZA REAL SUITE 275</rptOwnerStreet1>
            <rptOwnerStreet2></rptOwnerStreet2>
            <rptOwnerCity>BOCA RATON</rptOwnerCity>
            <rptOwnerState>FL</rptOwnerState>
            <rptOwnerZipCode>33432</rptOwnerZipCode>
            <rptOwnerStateDescription></rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>1</isDirector>
            <isOfficer>0</isOfficer>
            <isTenPercentOwner>0</isTenPercentOwner>
            <isOther>0</isOther>
            <officerTitle></officerTitle>
            <otherText></otherText>
        </reportingOwnerRelationship>
    </reportingOwner>

    <aff10b5One>0</aff10b5One>

    <nonDerivativeTable>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Common Stock</value>
            </securityTitle>
            <transactionDate>
                <value>2026-07-01</value>
            </transactionDate>
            <deemedExecutionDate></deemedExecutionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>A</transactionCode>
                <equitySwapInvolved>0</equitySwapInvolved>
            </transactionCoding>
            <transactionTimeliness></transactionTimeliness>
            <transactionAmounts>
                <transactionShares>
                    <value>30000</value>
                    <footnoteId id="F1"/>
                </transactionShares>
                <transactionPricePerShare>
                    <value>0</value>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>A</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>55000</value>
                    <footnoteId id="F2"/>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
    </nonDerivativeTable>

    <footnotes>
        <footnote id="F1">The reported transaction involved the Reporting Person's receipt of 30,000 restricted share units (&quot;RSUs&quot;). The RSUs were granted pursuant to the applicable RSU agreement and the Lixte Biotechnology Holdings, Inc. 2020 Stock Incentive Plan. Each RSU represents a contingent right to receive one share of common stock upon vesting, subject to continued service. The 30,000 RSUs vested upon the achievement of the performance conditions set forth in the Reporting Person's RSU agreement, which have been satisfied.</footnote>
        <footnote id="F2">The total reported in Column 5 includes (i) the 30,000 newly awarded RSUs, and (ii) 25,000 RSUs, all of which have vested.</footnote>
    </footnotes>

    <ownerSignature>
        <signatureName>/s/ Lourdes Felix</signatureName>
        <signatureDate>2026-07-02</signatureDate>
    </ownerSignature>
</ownershipDocument>
