HIV-associated neurocognitive disorders (HAND) affect up to 50% of HIV-infected adults despite potent viral suppression with antiretroviral therapy (ART) and are associated with persistent neuronal damage, monocyte/macrophage activation, chronic inflammation, and oxidative stress. Heme oxygenase-1 (HO-1) is a highly inducible, detoxifying enzyme critical for limiting oxidative stress, inflammation, and cellular injury within the central nervous system (CNS) and other tissues. Our analysis of HO-1 expression in the brain prefrontal cortex from HIV-infected individuals demonstrated a significant HO-1 protein deficiency, even in HIV-infected subjects treated with ART. This HO-1 deficiency associated with a diagnosis of HAND and HIV-encephalitis (HIVE) as well as with elevated CNS HIV replication, type I interferon responses, and macrophage activation. Within this cohort longer variants of a HO-1 promoter region (GT)n microsatellite polymorphism, which cause reduced HO-1 gene expression, associated with increased risk of HIVE and elevated CNS macrophage activation. HIV replication in macrophages, a primary CNS HIV reservoir, selectively reduced HO-1 protein and RNA expression and induced production of neurotoxic levels of glutamate. This HO-1 deficiency and associated neurotoxin production was a conserved feature of infection with macrophage-tropic HIV-1 and HIV-2 strains that correlated closely with the extent of replication. ART regimens applied to macrophages after HIV infection was established failed to prevent this HO-1 loss and associated neurotoxin production. HO-1 siRNA knockdown and enzymatic inhibition in HIV-infected macrophages increased supernatant glutamate and neurotoxicity. In contrast, increasing HO-1 expression through siRNA derepression or with pharmacologic inducers, including the CNS-penetrating drug dimethyl fumarate (DMF), decreased supernatant glutamate and neurotoxicity. These findings identify HO-1 as a host factor that is deficient in the brains of HIV-infected individuals and suggest that loss of HO-1 and its protective functions may contribute to HIV neuropathogenesis. Moreover, this work defines a predictable and conserved relationship between HIV replication, HO-1 loss, and neurotoxin production in macrophages that likely reflects processes in place in the HIV-infected brain of individuals receiving ART. Correcting this HO-1 deficiency could provide a novel approach for neuroprotection in individuals with or at risk for developing HAND above that provided by current ART.