Evodiamine is an alkaloidal compound with antiobesity 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 antiobesity effect of evodiamine could be attributed to the involvement of UCP1. When UCP1-knockout mice were fed a high-fat diet with 0.03% evodiamine (wt/wt) 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 ERK/MAPK, reduced the expression of transcription factors such as peroxisome proliferator-activated receptor-gamma, 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/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-knockout 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.