Vγ9Vδ2 T lymphocytes, a major γδ T lymphocyte subset in humans, display cytolytic activity against various tumor cells upon recognition of yet uncharacterized structures. Here, we show that an entity related to the mitochondrial F1-ATPase is expressed on tumor cell surface and promotes tumor recognition by Vγ9Vδ2 T cells. When immobilized, purified F1-ATPase induces selective activation of this lymphocyte subset. The Vγ9Vδ2 T cell receptors (TCR) and the F1-ATPase also bind a delipidated form of apolipoprotein A-I (apo A-I), as demonstrated by surface plasmon resonance. Moreover, the presence of apo A-I in the culture medium is required for optimal activation of Vγ9Vδ2 T cells by tumors expressing F1-ATPase. This study thus describes an unanticipated tumor recognition mechanism by Vγ9Vδ2 lymphocytes and a possible link between γδ T cell immunity and lipid metabolism.