Abstract Pentamethylquercetin (PMQ) is a natural quercetin derivative found in a variety of edible herb. Although PMQ has potential as anti-diabetic agent, there have been no reports on its anti-adipogenic effects in the obese animals. This study investigated whether PMQ attenuates high-fat diet (HFD)-induced adipogenesis in the epididymal fat tissues of mice and explored its underlying mechanisms. In comparison with HFD-fed mice, mice fed with PMQ showed significantly lower body weight gain, adipose tissue mass, and plasma levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol, and glucose, but higher plasma levels of high-density lipoprotein cholesterol. PMQ significantly reversed the HFD-induced regulation of Sirt1/mTOR signaling genes (Sirt1, mTOR, 4EBP1, and S6K1), and key adipogenic genes (PPARγ, SREBP1, FAS, ATGL, HSL, and Perilipin) in the epididymal adipose tissues of obese mice. However, nicotinamide appeared to partly inhibit PMQ-mediated anti-adipogenic effects involved in this attenuation. These results suggested that PMQ inhibited visceral adipogenesis by suppressing the Sirt1-mediated mTOR and adipogenesis signaling cascades. It might be a potential candidate for the treatment of obesity.