Mitochondrial dysfunction has been associated with long-term toxicities of human immunodeficiency virus (HIV) therapy, particularly with the nucleoside analog reverse transcriptase inhibitors (NRTIs). Lactic acidosis, hepatic steatosis, myopathies, cardiomyopathies, neuropathies, and lipodystrophy are frequently attributed to mitochondrial toxicity. Since mitochondrial toxicity could pose a major threat to the long-term success of HIV therapy, the scientific evidence underlying an association between mitochondrial toxicity and antiretroviral therapies, must be carefully examined. There is some data to support the association between NRTIs and mitochondria dysfunction. In this review, we examine human, animal, and in vitro data implicating mitochondrial dysfunction as the causal mechanism of NRTI-associated toxicity in HIV-infected patients.