•The CYSLTR1 antagonist montelukast protects neurons from macrophage neurotoxicity induced by HIV-1 or HIVgp120 protein.•HIV-1 infection or exposure to HIVgp120 increases CysLT release by macrophages and inhibition of p38 MAPK abrogates elevated CysLT production.•Cerebral cortex of HIV+ individuals with pathology expresses more CYSLTR1 than that of HIV+ persons without pathology or uninfected individuals.•Genetic ablation of Ltc4s or Cysltr1 in HIVgp120-transgenic mice results in complete neuroprotection and preserved memory function.•Genetic ablation of Ltc4s or Cysltr1 in mice reveals a physiological role of CysLTs in memory function and sexual dimorphisms in glial cells. Macrophages (MΦ) infected with human immunodeficiency virus (HIV)-1 or activated by its envelope protein gp120 exert neurotoxicity. We found previously that signaling via p38 mitogen-activated protein kinase (p38 MAPK) is essential to the neurotoxicity of HIVgp120-stimulated MΦ. However, the associated downstream pathways remained elusive. Here we show that cysteinyl-leukotrienes (CysLT) released by HIV-infected or HIVgp120 stimulated MΦ downstream of p38 MAPK critically contribute to neurotoxicity. SiRNA-mediated or pharmacological inhibition of p38 MAPK deprives MΦ of CysLT synthase (LTC4S) and, pharmacological inhibition of the cysteinyl-leukotriene receptor 1 (CYSLTR1) protects cerebrocortical neurons against toxicity of both gp120-stimulated and HIV-infected MΦ. Components of the CysLT pathway are differentially regulated in brains of HIV-infected individuals and a transgenic mouse model of NeuroHIV (HIVgp120tg). Moreover, genetic ablation of LTC4S or CysLTR1 prevents neuronal damage and impairment of spatial memory in HIVgp120tg mice. Altogether, our findings suggest a novel critical role for cysteinyl-leukotrienes in HIV-associated brain injury.