In diabetes mellitus, the excessive rate of glucose production from the liver is considered a primary contributor for the development of hyperglycemia, in particular, fasting hyperglycemia. In this study, we investigated whether kaempferol, a flavonol present in several medicinal herbs and foods, can be used to ameliorate diabetes in an animal model of insulin deficiency and further explored the mechanism underlying the anti-diabetic effect of this flavonol. We demonstrate that oral administration of kaempferol (50 mg/kg/day) to streptozotocin-induced diabetic mice significantly improved hyperglycemia and reduced the incidence of overt diabetes from 100% to 77.8%. This outcome was accompanied by a reduction in hepatic glucose production and an increase in glucose oxidation in the muscle of the diabetic mice, whereas body weight, calorie intake, body composition, and plasma insulin and glucagon levels were not altered. Consistently, treatment with kaempferol restored hexokinase activity in the liver and skeletal muscle of diabetic mice while suppressed hepatic pyruvate carboxylase activity and gluconeogenesis. These results suggest that kaempferol may exert antidiabetic action via promoting glucose metabolism in skeletal muscle and inhibiting gluconeogenesis in the liver.