Fibroblast growth factor 21 (FGF21), which structurally belongs to the FGF superfamily, acts as an endocrine factor and plays important roles in the regulation of energy homeostasis, glucose and lipid metabolism. The therapeutic administration of FGF21 analogs improves nonalcoholic steatohepatitis, the severe stage of non-alcoholic fatty liver disease, which is associated with obesity, metabolic syndrome, dyslipidemia and type 2 diabetes. In the present study, we screened crude extracts from 88 herbal drugs frequently used in Kampo prescriptions as potential anti-obesity agents by monitoring the production of FGF21 in C2C12 myotubes. Among these extracts screened, we found that 3-O-acetyloleanolic acid, an active constituent isolated from the fruits of Forsythia suspensa, stimulated FGF21 production in C2C12 myotubes. Additionally, significant increases in CRE-dependent luciferase activity were observed in cells overexpressing  bile acid receptor TGR5 in response to 3-O-acetyloleanolic acid treatment, which indicated that the responses caused by 3-O-acetyloleanolic acid was dependent on TGR5 activation. We observed that the phosphorylation of p38 was increased by 3-O-acetyloleanolic acid rapidly in C2C12 myotubes. Pretreatment with the selective p38 inhibitor SB203580 also significantly repressed the stimulatory effect of 3-O-acetyloleanolic acid on FGF21 secretion. These findings collectively indicated that TGR5 receptor signaling up-regulates FGF21 expression via p38 activation.