Background <p>TCRαβ⁺ double negative T cells (DNT) have recently gained attention for their antitumor activity. Adoptive DNT therapy has emerged as a promising cancer immunotherapy due to its potent cytotoxic function and lack of graft-versus-host-disease. However, the intrinsic mechanisms regulating DNT antitumor functions remain unclear.</p> Methods <p>Signaling lymphocytic activation molecule factor 7 (SLAMF7) expression in murine and human DNT were evaluated. The antitumor activities were compared between SLAMF7⁺ and SLAMF7⁻ DNT both <i>in vivo</i> and <i>in vitro</i>. Further, metabolomics analysis was performed to reveal the underlying mechanism by which SLAMF7 promotes DNT antitumor cytotoxicity.</p> Results <p>The expression of SLAMF7 was markedly increased on DNT upon activation. SLAMF7⁺ DNT exhibited superior antitumor capacity both <i>in vitro</i> and <i>in vivo</i> compared with SLAMF7⁻ DNT. Mechanistically, SLAMF7 enhanced antitumor activity through ligand-independent and ligand-dependent dual manners. Firstly, SLAMF7 could upregulate GPT2/SLC1A5-mediated glutamine metabolism by activating ERK signaling pathway in DNT, thereby supporting mitochondrial fitness, increasing ATP production, enhancing the expression of effector molecules such as granzyme B and perforin, and promoting antitumor activity of DNT against tumor cells independent of homotypic ligand-receptor interactions. Secondly, DNT showed superior antitumor cytotoxicity against SLAMF7-expressing tumor cells because SLAMF7-SLAMF7 interaction between DNT and SLAMF7-expressing tumor cells promoted DNT cell degranulation. Furthermore, SLAMF7 was also highly expressed in human DNT, and its dual antitumor roles in human DNT were also validated.</p> Conclusions <p>SLAMF7 is a key regulator of DNT-mediated cytotoxicity and a promising target for improving DNT cell function in cancer therapy.</p>