Evodiamine is an alkaloidal compound with anti-obesity effects that have been thought to be due to uncoupling protein-1 (UCP1) thermogenesis similar to the effects of capsaicin, but the underlying mechanisms are poorly understood. To clarify the mechanisms, we first examined whether the anti-obesity effect of evodiamine could be attributed to the involvement of UCP1. When UCP1-KO mice were fed a high fat diet with 0.03% evodiamine (w/w) for 2 months, the increases in body weight, adiposity, and the serum levels of leptin and insulin were reduced in a manner indistinguishable from control mice fed a high fat diet with evodiamine, suggesting that evodiamine triggered a UCP1-independent mechanism to prevent diet-induced obesity. By using preadipocyte cultures, we found that evodiamine, but not capsaicin, increased phosphorylation of extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinases (MAPK), reduced the expression of transcription factors such as peroxisome proliferator-activated receptor , and strongly inhibited adipocyte differentiation. Evodiamine treatment also reduced insulin-stimulated phosphorylation of Akt, a crucial regulator of adipocyte differentiation; and the reduction of phosphorylated-Akt and augmentation of phosphorylated-ERK were reversed by blockade of the MAPK kinase (MEK)/MAPK signaling pathway, restoring adipogenesis in the cultures. The changes in ERK and Akt phosphorylation levels were also observed in white adipose tissues of UCP1-KO mice fed the evodiamine diet. These findings suggest that evodiamine has a potential to prevent the development of diet-induced obesity in part by inhibiting adipocyte differentiation through ERK activation and its negative cross-talk with the insulin signaling pathway.