The lentiviral protein Nef plays a major role in the pathogenesis of human immunodeficiency virus type I (HIV-1) infection. Although the exact mechanisms of its actions are not fully understood, Nef has been shown to be essential for the maintenance of high-titer viral replication and disease pathogenesis in in vivo models of simian immunodeficiency virus infection of monkeys. Nef has also been suggested to play a pivotal role in the depletion of T cells by promoting apoptosis in bystander cells. In this context, we investigated the ability of extracellular and endogenously expressed HIV-1 Nef to induce apoptosis in primary human brain microvascular endothelial cells (MVECs). Human brain MVECs were exposed to baculovirus-expressed HIV-1 Nef protein, an HIV-1-based vector expressing Nef, spleen necrosis virus (SNV)-Nef virus (i.e., SNV vector expressing HIV-1 Nef as a transgene), and the HIV-1 strain ADA and its Nef deletion mutant, ADADeltaNef. We observed that ADA Nef, the HIV-1 vector expressing Nef, and SNV-Nef were able to induce apoptosis in a dose-dependent manner. The mutant virus with a deletion in Nef was able to induce apoptosis in MVECs to modest levels, but the effects were not as pronounced as with the wild-type HIV-1 strain, ADA, the HIV-1-based vector expressing Nef, or SNV-Nef viruses. We also demonstrated that relatively high concentrations of exogenous HIV-1 Nef protein were able to induce apoptosis in MVECs. Gene microarray analyses showed increases in the expression of several specific proapoptotic genes. Western blot analyses revealed that the various caspases involved with Nef-induced apoptosis are processed into cleavage products, which occur only during programmed cell death. The results of this study demonstrate that Nef likely contributes to the neuroinvasion and neuropathogenesis of HIV-1, through its effects on select cellular processes, including various apoptotic cascades.