A significant number of patients infected with human immunodeficiency virus-1 (HIV-1) suffer cognitive impairment ranging from mild to severe HIV-associated dementia (HAD), a result of neuronal degeneration in the basal ganglia, cerebral cortex and hippocampus. Mononuclear phagocyte dysfunction is thought to play an important role in the pathogenesis of HAD. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from over-stimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondrial glutaminase in HIV-infected macrophages is involved in converting glutamine to glutamate. Our results demonstrate that the concentration of glutamate in HIV-1 infected conditioned media was dependent on glutamine dose, and HIV-1 infected conditioned medium mediated glutamine-dependent neurotoxicity. These results indicate HIV-infection mediates neurotoxicity through glutamate production. In addition, glutamate-mediated neurotoxicity correlated with caspase activation and neuronal cell cycle re-activation. Inhibition of mitochondrial glutaminase diminished the HIV-induced glutamate production, and attenuated NMDA over-stimulation and subsequent neuronal apoptosis. These data implicate mitochondrial glutaminase in the induction of glutamate-mediated neuronal apoptosis during HIV-associated dementia, and provides a possible therapeutic strategy for HAD treatment.