Gamma delta (γδ) T cells are a unique subset of T cells crucial for various immune responses and immunopathology. The γδ T cell receptor (TCR) recognizes a diverse range of antigens independently of the major histocompatibility complex and associates with CD3 subunits to initiate T cell activation. This study reports the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR–CD3 complexes, revealing distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR–CD3 complex is monomeric with significant conformational flexibility in the extracellular domains and connecting peptides, while the Vγ5Vδ1 TCR–CD3 complex displays a dimeric architecture. The dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. The study also identifies a cholesterol-like molecule that wedges into the transmembrane region of the Vγ9Vδ2 TCR–CD3 complex, exerting an inhibitory role in TCR signaling. These findings provide insights into the unique properties of γδ TCR and facilitate immunotherapeutic interventions.Gamma delta (γδ) T cells are a unique subset of T cells crucial for various immune responses and immunopathology. The γδ T cell receptor (TCR) recognizes a diverse range of antigens independently of the major histocompatibility complex and associates with CD3 subunits to initiate T cell activation. This study reports the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR–CD3 complexes, revealing distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR–CD3 complex is monomeric with significant conformational flexibility in the extracellular domains and connecting peptides, while the Vγ5Vδ1 TCR–CD3 complex displays a dimeric architecture. The dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. The study also identifies a cholesterol-like molecule that wedges into the transmembrane region of the Vγ9Vδ2 TCR–CD3 complex, exerting an inhibitory role in TCR signaling. These findings provide insights into the unique properties of γδ TCR and facilitate immunotherapeutic interventions.