Antiretroviral therapy (ART) containing integrase strand transfer inhibitors (INSTIs) has been associated with weight gain in both ART initiation and switch studies, especially in women, but the underlying mechanisms are unclear. The effects of dolutegravir (DTG) on food intake, energy expenditure, oxygen consumption in female mice, and gene expression from adipose tissues were assessed. Human and murine preadipocytes were treated with DTG either during differentiation into mature brown/beige adipocytes or postdifferentiation. Lipid accumulation, lipolysis, β-adrenergic response, adipogenic markers, mitochondrial respiration, and insulin response were analyzed. Two-week administration of DTG to female mice reduced energy expenditure, which was accompanied by decreased uncoupling protein 1 (UCP1) expression in brown/beige adipose tissues. In vitro studies showed that DTG significantly reduced brown adipogenic markers, especially UCP1 in brown and beige adipocytes, whereas drugs from other classes did not. Furthermore, a loss of UCP1 by DTG led to a decrease in mitochondrial complex IV component, followed by a reduction in mitochondrial respiratory capacity and reduced insulin-stimulated glucose uptake. Our findings show that DTG targets UCP1 and mitochondrial functions in brown and beige adipocytes and disrupts thermogenic functions in preclinical models, providing the potential mechanisms by which DTG suppresses energy expenditure leading to weight gain.