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

    <schemaVersion>X0508</schemaVersion>

    <documentType>4</documentType>

    <periodOfReport>2025-12-21</periodOfReport>

    <issuer>
        <issuerCik>0001713863</issuerCik>
        <issuerName>Rafael Holdings, Inc.</issuerName>
        <issuerTradingSymbol>RFL</issuerTradingSymbol>
    </issuer>

    <reportingOwner>
        <reportingOwnerId>
            <rptOwnerCik>0001726896</rptOwnerCik>
            <rptOwnerName>Polinsky David</rptOwnerName>
        </reportingOwnerId>
        <reportingOwnerAddress>
            <rptOwnerStreet1>C/O RAFAEL HOLDINGS, INC.</rptOwnerStreet1>
            <rptOwnerStreet2>520 BROAD ST</rptOwnerStreet2>
            <rptOwnerCity>NEWARK</rptOwnerCity>
            <rptOwnerState>NJ</rptOwnerState>
            <rptOwnerZipCode>07102</rptOwnerZipCode>
            <rptOwnerStateDescription></rptOwnerStateDescription>
        </reportingOwnerAddress>
        <reportingOwnerRelationship>
            <isDirector>false</isDirector>
            <isOfficer>true</isOfficer>
            <isTenPercentOwner>false</isTenPercentOwner>
            <isOther>false</isOther>
            <officerTitle>Chief Financial Officer</officerTitle>
        </reportingOwnerRelationship>
    </reportingOwner>

    <aff10b5One>false</aff10b5One>

    <nonDerivativeTable>
        <nonDerivativeTransaction>
            <securityTitle>
                <value>Class B Common Stock, par value $.01 per share</value>
            </securityTitle>
            <transactionDate>
                <value>2025-12-21</value>
            </transactionDate>
            <transactionCoding>
                <transactionFormType>4</transactionFormType>
                <transactionCode>F</transactionCode>
                <equitySwapInvolved>false</equitySwapInvolved>
            </transactionCoding>
            <transactionAmounts>
                <transactionShares>
                    <value>1961</value>
                    <footnoteId id="F1"/>
                </transactionShares>
                <transactionPricePerShare>
                    <value>1.285</value>
                </transactionPricePerShare>
                <transactionAcquiredDisposedCode>
                    <value>D</value>
                </transactionAcquiredDisposedCode>
            </transactionAmounts>
            <postTransactionAmounts>
                <sharesOwnedFollowingTransaction>
                    <value>258680</value>
                    <footnoteId id="F2"/>
                </sharesOwnedFollowingTransaction>
            </postTransactionAmounts>
            <ownershipNature>
                <directOrIndirectOwnership>
                    <value>D</value>
                </directOrIndirectOwnership>
            </ownershipNature>
        </nonDerivativeTransaction>
    </nonDerivativeTable>

    <footnotes>
        <footnote id="F1">Represents shares withheld by the Issuer for tax purposes upon the vesting of Restricted Stock.</footnote>
        <footnote id="F2">Consists of 76,882 shares held directly, 86,798 fully vested restricted shares and 95,000 unvested restricted shares that vest as follows: 6,250 quarterly from March 21, 2026 through December 21, 2026; 10,000 on each of October 25, 2026 and October 25, 2027; and 12,500 on each of January 13, 2026, January 13, 2027, January 13, 2028 and January 13, 2029.</footnote>
    </footnotes>

    <ownerSignature>
        <signatureName>Joyce J. Mason, by Power of Attorney</signatureName>
        <signatureDate>2025-12-23</signatureDate>
    </ownerSignature>
</ownershipDocument>
