The HIV-1 accessory proteins Nef and Vpu alter cell surface levels of multiple host proteins to modify the immune response and increase viral persistence. Nef and Vpu can downregulate cell surface levels of the co-stimulatory molecule CD28, however the mechanism of this function has not been completely elucidated. Here, we provide evidence that Nef and Vpu decrease cell surface and total cellular levels of CD28. Moreover, using inhibitors we implicate the cellular degradation machinery in the downregulation of CD28. We shed light on the mechanisms of CD28 downregulation by implicating the Nef LL165 and DD175 motifs in decreasing cell surface CD28 and Nef DD175 in decreasing total cellular CD28. Moreover, the Vpu LV64 and S52/56 motifs were required for cell surface CD28 downregulation, while, unlike for CD4 downregulation, Vpu W22 was dispensable. The Vpu S52/56 motif was also critical for Vpu-mediated decreases in total CD28 protein level. Finally, the ability of Vpu to downregulate CD28 is conserved between multiple group M Vpu proteins and infection with viruses encoding or lacking Nef and Vpu have differential effects on activation upon stimulation. We report that Nef and Vpu downregulate cell surface and total cellular CD28 levels. We identified inhibitors and mutations within Nef and Vpu that disrupt downregulation, shedding light on the mechanisms utilized to downregulate CD28. The conservation and redundancy between the abilities of two HIV-1 proteins to downregulate CD28 highlight the importance of this function, which may contribute to the development of latently infected cells.