Objective To establish a stable and reliable mouse model as an alternative to the traditional model of impaired glucose tolerance induced by calorie restriction and its effect on glucose homeostasis. Methods Forty 16-week-old SPF C57BL/6J mice (half male and half female) were randomly divided into four groups by sex and the way of feeding. The mice in the ad libitum (AL) group had free access to basic diet, while the mice in the intermittent fasting (IF) group had normal diet and fasting on alternate days, with free access to water on the fasting days. The changes of body weight and blood glucose concentration in each group were monitored, and intraperitoneal glucose tolerance test and insulin tolerance test in mice were performed before and after the 12-week IF treatment. Results At 12 weeks after IF treatment, the body weight and blood glucose concentration of mice did not show significant difference. After i.p. injection of glucose, the blood glucose concentration of IF mice was less increased than the AL group, and after the insulin injection, the blood glucose concentration was more decreased. Compared to the AL group, the areas under the curve of tolerance test in the IF group were significantly decreased (P< 0.05). Conclusions After IF treatment, the mice show an enhanced sensitivity to insulin and improved glucose tolerance. This establishment method of mouse model of intermittent fasting is easy and simple, therefore, can be used as an effective alternative to traditional calorie restriction model of impaired glucose tolerance.