The antidiabetic activity of Lyophyllum decastes (Tricholomataceae) was investigated in KK-Ay mice, an animal model of genetically type 2 diabetes with hyperinsulinemia. The water extract of Lyophyllum decastes (LD) (500 mg/kg body weight) reduced the blood glucose of KK-Ay mice 7 h after a single oral administration (p<0.05) when compared with control. LD reduced the blood glucose of KK-Ay mice 3 weeks after repeated administration (p<0.05), and also significantly lowered the serum insulin of KK-Ay mice under similar conditions (p<0.01). However, LD did not affect the blood glucose in normal mice. LD tended to decrease of the blood glucose in an insulin tolerance test. In addition, the muscle content of facilitative glucose transporter isoform 4 (GLUT4) protein content in the plasma membrane fraction from muscle significantly increased in the orally LD-treated KK-Ay mice when compared to that of the controls (p<0.01). These results suggest that the antidiabetic activity of LD is derived, at least in part, from a decrease in insulin resistance, due to the increase of GLUT4 protein content in the plasma membrane of the muscle.