Increasing energy expenditure through activation of brown adipose tissue (BAT) is a critical approach to treating obesity and diabetes. In this study, rutin, a natural compound extracted from mulberry and a drug used as a capillary stabilizer clinically for many years without any side effects, regulated whole-body energy metabolism by enhancing BAT activity. Rutin treatment significantly reduced adiposity, increased energy expenditure, and improved glucose homeostasis in both genetically obese (Db/Db) and diet-induced obesity (DIO) mice. Rutin also induced brown-like adipocyte (beige) formation in subcutaneous adipose tissue in both obesity mouse models. Mechanistically, we found that rutin directly bound to and stabilized SIRT1, leading to hypoacetylation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha protein, which stimulated Tfam transactivation and eventually augmented the number of mitochondria and UCP1 activity in BAT. These findings reveal that rutin is a novel small molecule that activates BAT and may provide a novel therapeutic approach to the treatment of metabolic disorders.-Yuan, X., Wei, G., You, Y., Huang, Y., Lee, H. J., Dong, M., Lin, J., Hu, T., Zhang, H., Zhang, C., Zhou, H., Ye, R., Qi, X., Zhai, B., Huang, W., Liu, S., Xie, W., Liu, Q., Liu, X., Cui, C., Li, D., Zhan, J., Cheng, J., Yuan, Z., Jin, W. Rutin ameliorates obesity through brown fat activation.