Objective Oral glucose tolerance testing (OGTT) is a method used to study the function of islet β- cells and blood glucose regulation. Fasting is necessary beforehand, but fasting and glucose-stimulated blood glucose concentrations and insulin concentrations differ according to the duration of fasting. This study aimed to identify the optimal fasting time prior to OGTT in the widely used KM mouse strain. Methods Clean grade male KM male mice aged 6-7 weeks were randomly allocated to four groups. After fasting for 4, 8, 10, or 12 h, OGTT was performed, blood glucose and insulin concentrations were measured, and the concentration vs. time curves for each were compared. Results 1) The blood glucose and insulin concentrations of all the mice increased from their fasting values to a peak, and then decreased back to the baseline values. Compared with the fasting blood glucose concentration of mice that were fasted for 4 h, the fasting blood glucose concentrations of the other three groups were significantly lower ( P < 0. 01). (2) The blood glucose concentrations in the 4 h-fasted group were also the highest at each subsequent time point, and the area under the blood glucose curve was significantly different from those in the other groups ( P < 0. 05). (3)The fasting insulin concentration in the 4 h-fasted group was 4% higher than that of the 8 h-fasted group, which had a value about twice as high as the 12 hfasted group. The area under the insulin curve was lowest in the mice fasted for 8 h. Conclusions KM mice fasted for 8- 10 h prior to OGTT showed large differences in blood glucose and insulin concentrations, higher resolution, and larger changes in values. This made the arrangement of fasting time more reasonable, and the experimental plan is reasonable and efficient. When KM mice are used to establish a diabetes model, the effect of fasting time prior to OGTT should be considered.