Myricetin is a biologically active natural polyphenol with beneficial effects on metabolic health. This study aimed to examine the effects of myricetin on the expression levels of genes involved in lipolysis and mitochondrial respiration in adipocytes and the anti-obesity potential of myricetin. The results indicated that myricetin reduced triglyceride (TG) content and increased mitochondrial content and oxygen consumption rate (OCR) in adipocytes in vitro. To determine anti-obesity effect of myricetin, C57BL6/J mice were fed a high-fat diet (HFD) for eight weeks and then treated with myricetin (10 mg/kg) for 2 weeks. The in vivo treatment of myricetin reduced body weight by 11%. Furthermore, it improved the glucose tolerance, and increased fatty acid consumption of HFD-fed mice. Myricetin treatment increased Sirt3 expression and reduced the acetylation of mitochondrial proteins in adipose tissue. Finally, the knockdown of Sirt3 in adipocytes reduced the myricetin-induced increase in mitochondrial oxygen consumption rate by about 27% compared to controls. Our results indicated that myricetin exerted anti-obesity effects through the upregulation of Sirt3 expression and mitochondrial metabolism in adipose tissue.