Despite the benefits of antiretroviral therapy (ART), a large percentage of people with HIV (PWH) still suffer from some form of neurocognitive impairment. Studies have shown that high degree of oxidative stress and inflammation remain present in the central nervous system of PWH, which can activate the integrated stress response (ISR) pathway. We previously showed that the levels of several markers for ISR activation, such as phosphorylated eukaryotic initiation factor 2α (P-eIF2α), were elevated in neurons and astrocytes in the cortex in autopsy brain tissue of PWH. Phosphorylation of eIF2α is mediated by four kinases, which will result in ISR activation, as reported in several neurodegenerative disorders. In general, ISR is an adaptive pathway; however, chronic ISR activation may contribute to neuronal damage and neurocognitive impairment in PWH. Recently, several studies have reported that certain ART drugs contribute to the persistence of HIV-associated neurocognitive disorders and can induce the ISR. The aim of the present study was to assess ISR activation in neurons, astrocytes, and oligodendrocytes in brain tissue samples of SIV-infected rhesus macaques in a lentiviral model of HIV infection. Methods We examined necropsy brain tissue specimens of 11 rhesus macaques, including SIV-infected/ART-untreated macaques (n = 3), SIV-infected/ART-treated macaques (n = 4), and uninfected/untreated (n = 4) macaques, with the aim to determine if ART aggravated ISR activation in the CNS. Formalin-fixed/paraffin-embedded sections of cortical tissue were immunofluorescently stained using an antibody to p-eIF2α to assess ISR activation and antibodies against MAP2, GFAP, and ASPA to label neurons, astrocytes, and oligodendrocytes, respectively. Results By semiquantitative analysis of images of the stained specimens we found that p-eIF2α levels in neurons was significantly higher in the SIV-infected/ART-treated group than in the SIV-infected/ART-untreated group. However, we did not observe differences in p-eIF2α levels in astrocytes the gray matter and oligodendrocytes in the white matter among the three groups. Conclusion In our study, we observed a significant increase in ISR activation in neurons in the gray matter of the SIV-infected/ART-treated rhesus macaques compared to the SIV-infected/ART-untreated rhesus macaques, which we did not observe in astrocytes and oligodendrocytes.