Mulberry is one of the Chinese traditional medicines. But few studies focused on the hypoglycemic effect of Mulberry seed and the possible mechanisms. We identified that mulberry seed is rich in DNJ, the content of which is 0.6015%. Both mulberry seeds powder and its aqueous extract were used to detect the hypoglycemic effect of mulberry seeds. The results indicated that mulberry seeds powder could reduce the FBG, PBG and HbA1c, and abate the organ injuries such as liver, kidney and spleen of diabetic mice. The results also showed that aqueous extract of mulberry seed powder orally to diabetic mice significantly improved the glucose tolerance of mice, and the mulberry seeds aqueous extract significantly inhibited the activity of sucrase and maltase in vitro. These suggested that Mulberry seeds perform high hypoglycemic effect by inhibition of α-glucosidase activity.