Human T cells that express a T cell antigen receptor (TCR) containing gamma-chain variable region 9 and delta-chain variable region 2 (Vgamma9Vdelta2) recognize phosphorylated prenyl metabolites as antigens in the presence of antigen-presenting cells but independently of major histocompatibility complex (MHC), the MHC class I-related molecule MR1 and antigen-presenting CD1 molecules. Here we used genetic approaches to identify the molecule that binds and presents phosphorylated antigens. We found that the butyrophilin BTN3A1 bound phosphorylated antigens with low affinity, at a stoichiometry of 1:1, and stimulated mouse T cells with transgenic expression of a human Vgamma9Vdelta2 TCR. The structures of the BTN3A1 distal domain in complex with host- or microbe-derived phosphorylated antigens had an immunoglobulin-like fold in which the antigens bound in a shallow pocket. Soluble Vgamma9Vdelta2 TCR interacted specifically with BTN3A1-antigen complexes. Accordingly, BTN3A1 represents an antigen-presenting molecule required for the activation of Vgamma9Vdelta2 T cells.