Abstract Human immunodeficiency virus type 1 (HIV-1) Vpu is phosphorylated at two serine residues (Ser52 and Ser56) present within the acidic dodecapeptide region of the 54-aa cytoplasmic domain. Previous experiments have shown that Vpu phosphorylation is critical for the degradation of CD4 in the endoplasmic reticulum. In this study, we carried out experiments to elucidate the role of individual phosphoacceptor sites in CD4 proteolysis. We show here that acidic amino acids could not functionally substitute for phosphoserines in Vpu that is capable of inducing the degradation of CD4. Our studies have further revealed that phosphorylation of either of the two phosphoacceptor sites is not sufficient to generate a functional Vpu protein. When tested for functional complementation, inactive phosphorylation-proficient Vpu mutants failed to generate Vpu proteins that had the ability to induce the degradation of Vpu-sensitive glycoproteins. The failure to complement was not due to assembly defects in the Vpu protein as unphosphorylated Vpu formed oligomeric complexes in the cell. We also showed that Vpu expression inhibits protein transport in a phosphorylation-dependent manner. Our studies have thus revealed that both phosphoserines in Vpu are critical participants in a pathway that leads to the proteolysis of CD4 in the ER and that these phosphoserines should be present on the same subunit of the Vpu protein.