Previous studies have reported the anti-obesity effects of α, β-Amyrin in high fat-fed mice. This study aimed to evaluate whether α, β-Amyrin has an anti-adipogenic effect in 3T3-L1 murine adipocytes and to explore the possible underlying mechanisms. 3T3-L1 pre-adipocytes were differentiated in a medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Cytotoxicity of α, β-Amyrin was assessed by MTT assay. Lipid content in adipocytes was determined by Oil-Red O staining. In addition, the protein expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding proteins alpha (C/EBPα), beta (C/EBPβ), and delta (C/EBPδ) and glucose transporter 4 (GLUT4) were determined by qRT-PCR and western blot analysis. Oil-Red O staining revealed markedly reduced fat accumulation by α, β-Amyrin (6.25–50 μg/mL) without affecting cell viability. Furthermore, our results indicate that α, β-Amyrin can significantly suppress the adipocyte differentiation by downregulating the expression levels of adipogenesis-related key transcription factors such as PPARγ and C/EBPα, but not C/EBPβ or C/EPBδ. In addition, the protein expression of membrane GLUT4 in 3T3- L1 adipocytes treated with α, β-Amyrin was significantly higher than in control cells, indicating that α, β-Amyrin augments glucose uptake. These findings suggest that α, β-Amyrin exerts an anti-adipogenic effect principally via modulation of lipid and carbohydrate metabolism in 3T3-L1cells. The present in vitro findings, taken together with our earlier observation of the anti-obesity effect in vivo, suggest that α, β-Amyrin can be developed as a new therapeutic agent for treatment and prevention of obesity.