Vpu promotes the efficient release of human immunodeficiency virus type 1 (HIV-1) by overcoming the activity of tetherin, a host cell restriction factor that retains assembled virions at the cell surface. In this study, we analyzed the intracellular localization and trafficking of subtype B Vpu in HIV-1-producing human cells. We found that mutations of conserved positively charged residues (R30 and K31) within the putative overlapping tyrosine- and dileucine-based sorting motifs of the Vpu hinge region affected both the accumulation of the protein in the trans-Golgi network (TGN) and its efficient delivery to late endosomal degradative compartments. A functional characterization of this mutant revealed that the mislocalization of Vpu from the TGN correlated with an attenuation of HIV-1 release. Interestingly, clathrin light chain small interfering RNA-directed disruption of Vpu trafficking from the TGN to the endosomal system slightly stimulated Vpu-mediated HIV-1 release and completely restored the activity of the Vpu R30A,K31A mutant. An analysis of the C-terminal deletion mutants of Vpu identified an additional determinant in the second helical structure of the protein, which regulated TGN retention/localization, and further revealed the functional importance of Vpu localization in the TGN. Finally, we show that a large fraction of Vpu colocalizes with tetherin in the TGN and provide evidence that the degree of Vpu colocalization with tetherin in the TGN is important for efficient HIV-1 release. Taken together, our results reveal that Vpu traffics between the TGN and the endosomal system and suggest that the proper distribution of Vpu in the TGN is critical to overcome the restricting activity of tetherin on HIV-1 release.