Background: Obesity is defined as excessive fat mass and is a major cause of many chronic diseases such as diabetes, cardiovascular disease, and cancer. Increasing energy expenditure and regulating adipose tissue metabolism are important targets for the treatment of obesity. Serotonin (5-hydroxytryptophan [5-HT]) is a monoamine metabolite of the essential amino acid tryptophan. Here, we demonstrated that 5-HT in mature adipocytes regulated energy expenditure and lipid metabolism. Methods: Tiyptophan hydroxylase 1 (TPH1) is the rate-limiting enzyme during 5-HT synthesis in non-neural peripheral tissues. We generated adipose tissue-specific Tph1 knockout (Tph1 FKO) mice and adipose tissue-specific serotonin receptor 2A KO (Htr2a FKO) mice and analyzed their phenotypes during high-fat diet (HFD) induced obesity. Results: Tph1 FKO mice fed HFD exhibited reduced lipid accumulation, increased thermogenesis, and resistance to obesity. In addition Htr2a FKO mice fed HFD showed reduced lipid accumulation in white adipose tissue and resistance to obesity. Conclusion: These data suggest that the inhibition of serotonin signaling might be an effective strategy in obesity.