Browning white adipocytes may be a new target in anti-obesity therapy. Pentamethylquercetin (PMQ) has been shown to have anti-obesity effects in monosodium glutamate-induced obese mice. Here, we aimed to study the anti-obesity effects of PMQ in vitro and in vivo and to determine if adipose browning is involved in the mechanism underlying the anti-obesity effects of PMQ. We evaluated the effects of PMQ on cell proliferation, cell differentiation, glucose consumption, cellular lipid metabolism, and related brown gene expression in 3T3-L1 adipocytes. We also investigated the effects of PMQ in a mouse model of high-fat diet (HFD)-induced obesity. Our results demonstrated that PMQ increased the consumption of glucose, inhibited the accumulation of cellular triglycerides (TGs), and induced the expression of brown adipocyte-specific genes, such as uncoupling protein 1 (UCP-1), during the early stage of differentiation in 3T3-L1 adipocytes. In HFD mice, PMQ treatment reduced waist circumference, LEE index, white adipose tissue (WAT) weight and white adipocyte size and increased brown adipose tissue (BAT) weight. Moreover, PMQ treatment induced mitochondrial biogenesis and upregulated UCP-1 expression in WAT. These findings suggest that PMQ may induce browning of adipose tissue, a phenomenon that is at least partly related to its anti-obesity effects.