Cannabis use is highly prevalent especially among people living with HIV (PLWH). Activation of the anti-inflammatory and neuroprotective endocannabinoid system by phytocannabinoids, i.e. Δ9-tetrahydrocannabinol (THC), has been proposed to reduce HIV symptoms. However, THC’s effects on HIV-associated cognitive impairments are unclear. Using HIV-1 Tat transgenic mice, the current study investigates acute THC effects on various behavioral outcomes and the endocannabinoid system. Minor or no effects of THC doses (1, 3, 10 mg/kg) were noted for body mass, body temperature, locomotor activity, and coordination, but spontaneous nociception was significantly decreased, with Tat induction increasing antinociceptive THC effects. Anxiogenic effects of THC (10 mg/kg) were demonstrated in Tat(−) females and males compared to vehicle-treated mice, with overall increased anxiety-like behavior in females compared to males. Object recognition memory was diminished by acute THC (10 mg/kg) injections in Tat(−) but not Tat(+) females, without affecting males. For the endocannabinoid system and related lipids, no effects were noted for acute THC, but female sex and Tat induction was associated with elevated 2-AG, AEA, AA, CB1R, CB2R, FAAH and/or MAGL expression in various CNS regions. Further, females demonstrated higher AEA levels compared to males in most CNS structures, and AEA levels in the prefrontal cortex of Tat(+) females were negatively associated with recognition memory. Overall, findings indicate that acute THC exposure exerts differential effects on behavior in the context of neuroHIV dependent on sex, potentially due to an altered endocannabinoid system, which may be of relevance in view of potential cannabis-based treatment options for PLWH.