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Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design. • The crystal structure of HMBPP-bound intracellular BTN3A1 was determined at 1.97 Å • HMBPP forms hydrogen bonds with H 351 for efficient Vγ9Vδ2 T cell activation • An asymmetric intracellular dimer is involved in HMBPP-mediated γδ T cell activation • HMBPP doubles the binding force between extracellular BTN3A and Vγ9Vδ2 TCR Vγ9Vδ2 T cells sense tumor and microbial metabolites without MHC restriction. Yang et al. used a multifaceted approach to show how the highly potent microbial HMBPP binds to BTN3A1 and triggers inside-out signaling to activate Vγ9Vδ2 T cells. This study will have implications in emerging clinical applications of allogeneic Vγ9Vδ2 T cells. [ABSTRACT FROM AUTHOR]