Abstract Activation of mTORC1 is essential for anti-tumor function of iNKT cells. The mechanisms underlying impaired mTORC1 activation in intratumoral iNKT cells remain unclear. Here, we showed that lactic acid from tumor cells increased Vam6 expression in iNKT cells which led to impaired mTORC1 activation and IFN-γ production. Mechanistically, Vam6 in iNKT cells was essential for Rab7a-Vam6-AMPK complex formation and thus for recruitment of AMPK to lysosome to activate AMPK, a negative regulator of mTORC1. Additionally, Vam6 relieved inhibitory effect of VDAC1 on Rab7a-Vam6-AMPK complex formation at mitochondria-lysosome contact site. Given the key roles of lactic acid-increased Vam6 in promoting AMPK activation in intratumoral iNKT cells, reducing Vam6 expression significantly enhanced the mTORC1 activation in intratumoral iNKT cells as well as their anti-tumor efficacy. Together, we propose Vam6 as a target for iNKT cell-based immunotherapy.