The anti-tumor response of Vγ9Vδ2 T cells requires the sensing of accumulated phosphoantigens (pAgs) bound intracellularly to butyrophilin 3A1 (BTN3A1). In this study, we show that butyrophilin 2A1 (BTN2A1) is required for BTN3A-mediated Vγ9Vδ2 T cell cytotoxicity against cancer cells, and that expression of the BTN2A1/BTN3A1 complex is sufficient to trigger Vγ9Vδ2 TCR activation. Also, BTN2A1 interacts with all isoforms of BTN3A (BTN3A1, BTN3A2, BTN3A3), which appears to be a rate-limiting factor to BTN2A1 export to the plasma membrane. BTN2A1/BTN3A1 interaction is enhanced by pAgs and, strikingly, B30.2 domains of both proteins are required for pAg responsiveness. BTN2A1 expression in cancer cells correlates with bisphosphonate-induced Vγ9Vδ2 T cell cytotoxicity. Vγ9Vδ2 T cell killing of cancer cells is modulated by anti-BTN2A1 monoclonal antibodies (mAbs), whose action relies on the inhibition of BTN2A1 binding to the Vγ9Vδ2TCR. This demonstrates the potential of BTN2A1 as a therapeutic target and adds to the emerging butyrophilin-family cooperation pathway in γδ T cell activation. Display omitted •BTN2A1 expression in cancer cells correlates with Vγ9Vδ2 T cell cytotoxicity•BTN2A1 interacts with BTN3A1/3A2/3A3, leading to enhanced plasma membrane export•B30.2 domains of both BTN3A1 and BTN2A1 are required for pAg responsiveness•Anti-BTN2A1 mAbs blocking Vγ9Vδ2TCR binding antagonize Vγ9Vδ2 T cell cytotoxicity Cano et al. show that targeting BTN2A1 with mAbs allows modulation of Vγ9Vδ2 T cell cytotoxicity against primary cancer cells. BTN2A1 expression correlates with antitumoral Vγ9Vδ2 T cell responses. BTN2A1 export at the plasma membrane is strongly enhanced by interaction with BTN3A1, and this interaction is enhanced by amino-biphosphonate treatment.