The present study investigated the anti-diabetic activity and potential mechanism of stigmasterol (SMR), which is a kind of phytosterols derived from the edible soybean oil in vitro and in vivo. SMR displayed a mild GLUT4 translocation activity by 1.44-fold in L6 cells. L6 cells were treated with different concentration of SMR, showing significant effects on the enhancing glucose uptake. SMR administrated orally to the KK-Ay mice significantly alleviated their insulin resistance and oral glucose tolerance with reducing fasting blood-glucose levels and blood lipid indexes such as triglyceride and cholesterol. Moreover, the GLUT4 expression in L6 cells, skeletal muscle and white adipose tissue had been also enhanced. In this paper we conclude that, stigmasterol seems to have potential beneficial effects on the treatment of type 2 diabetes mellitus with the probable mechanism of targeting GLUT4 glucose transporter included increasing GLUT4 translocation and expression.