Carboxylesterase 3 (Ces3) is a hydrolase with a wide range of activities in liver and adipose tissue. In this study, we identified Ces3 as a major lipid droplet surface-targeting protein in adipose tissue upon cold exposure by liquid chromatography—tandem mass spectrometry. To investigate the function of Ces3 in the β-adrenergic signaling–activated adipocytes, we applied WWL229, a specific Ces3 inhibitor, or genetic inhibition by siRNA to Ces3 on isoproterenol (ISO)–treated 3T3-L1 and brown adipocyte cells. We found that blockage of Ces3 by WWL229 or siRNA dramatically attenuated the ISO-induced lipolytic effect in the cells. Furthermore, Ces3 inhibition led to impaired mitochondrial function measured by Seahorse. Interestingly, Ces3 inhibition attenuated an ISO-induced thermogenic program in adipocytes by downregulating Ucp1 and Pgc1α genes via peroxisome proliferator–activated receptor . We further confirmed the effects of Ces3 inhibition in vivo by showing that the thermogenesis in adipose tissues was significantly attenuated in WWL229-treated or adipose tissue–specific Ces3 heterozygous knockout (Adn-Cre-Ces3flx/wt) mice. As a result, the mice exhibited dramatically impaired ability to defend their body temperature in coldness. In conclusion, our study highlights a lipolytic signaling induced by Ces3 as a unique process to regulate thermogenesis in adipose tissue.