HIV-1 envelope spike (Env) is a type I membrane protein that mediates fusion of viral and cell membranes. We have determined an NMR structure at atomic resolution of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. It forms a well-ordered trimer that protects a conserved arginine, buried in the membrane. An N-terminal coiled coil and a C-terminal hydrophilic core stabilize the trimer; the latter may be structurally coupled to the cytoplasmic tail. Individual mutations of conserved residues do not completely disrupt the TM trimer, and they have minimal impact on membrane fusion and viral infectivity. Major changes in the hydrophilic core, however, can alter antibody sensitivity of the functional Env. These results show how a TM domain anchors, stabilizes and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design.