Inclusion of HIV-protease inhibitors in the treatment of people living with HIV⁺ has markedly decreased mortality but also increased the incidence of metabolic abnormalities, causes of which are not well understood. Here, we report that insulinopenia is exacerbated when Zucker fa/fa rats are exposed to a protease inhibitor for seven weeks, suggesting that chronic protease inhibitor exposure adversely affects pancreatic islet -cell function. In support of this possibility, we find increased apoptosis, as reflected by TUNEL fluorescence analyses, and reduced insulin secretory capacity in insulinoma cells and human pancreatic islet cells after in vitro exposures (48-96h) to clinically relevant protease inhibitors (ritonavir, lopinavir, atazanavir, or tipranavir). Further, pancreatic islets isolated from rats administered a HIV-protease inhibitor for three weeks exhibit greater cell death than islets isolated from vehicle-administered rats. The higher incidence of HIV- protease inhibitor-induced cell death was associated with cleavage and hence activation of caspase-3 and PARP but not with activation of PERK or induction of ER stress apoptotic factor CHOP. Exposure to the HIV-protease inhibitors, however, led to activation of mitochondria-associated caspase-9, caused a loss in mitochondrial membrane potential, and promoted the release of cytochrome c, suggesting that HIV-protease inhibitors currently in clinically use can induce -cell apoptosis by activating the mitochondrial apoptotic pathway. These findings therefore highlight the importance of considering -cell viability and function when assessing loss of glycemic control and the course of development of diabetes in HIV⁺ subjects receiving a protease inhibitor.