Tetherin (BST2/CD317) potently restricts the particle release of human immunodeficiency virus type 1 (HIV-1) mutants defective in the accessory gene vpu. Vpu antagonizes tetherin activity and induces its cell surface downregulation and degradation in a manner dependent on the transmembrane (TM) domains of both proteins. We have carried out extensive mutagenesis of the HIV-1 NL4.3 Vpu TM domain to identify three amino acid positions, A14, W22, and, to a lesser extent, A18, that are required for tetherin antagonism. Despite the mutants localizing indistinguishably from the wild-type (wt) protein and maintaining the ability to multimerize, mutation of these positions rendered Vpu incapable of coimmunoprecipitating tetherin or mediating its cell surface downregulation. Interestingly, these amino acid positions are predicted to form one face of the Vpu transmembrane alpha helix and therefore potentially contribute to an interacting surface with the transmembrane domain of tetherin either directly or by modulating the conformation of Vpu oligomers. While the equivalent of W22 is invariant in HIV-1/SIVcpz Vpu proteins, the positions of A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N, but not those from group O or SIVcpz that lack human tetherin (huTetherin)-antagonizing activity, suggesting that they may have contributed to the adaption of HIV-1 to human tetherin.