Objective To explore the optimal method of producing a streptozotocin-induced diabetic nephropathy C57BL/ 6 mice model by investigating a molded rate of single high-dose and multiple low-dose streptozotocin injections. Methods Male C57BL/ 6J mice ( n= 48) were randomly divided into three groups: the normal group and two diabetic nephropathy groups. Mice in one model group (the single high-dose group) were intraperitoneally administered 150 mg / kg streptozotocin after having a high fat / high sucrose diet. The other model group ( the multiple low-dose group ) was intraperitoneally administered 50 mg / kg streptozotocin for 4 consecutive days after having a high fat / high sucrose diet. The fasting blood glucose levels, body weight, water intake, 24 h urine protein, renal function and renal pathological changes of the three groups were measured. Results The body weight of the single high-dose group increased stably after streptozotocin injection, while the body weight of the multiple low-dose group decreased by inches after streptozotocin injection. Moreover, the fasting blood glucose levels, water intake and 24 h urine protein increased stably in the multiple low-dose group from week 2 to week 10. The levels of fasting blood glucose, 24 h urine protein, total cholesterol, triglycerides, low density lipoprotein, serum creatinine, blood urea nitrogen and kidney / body weight ratio were significantly higher in the multiple low-dose group than in the single high-dose group ( P< 0. 05) and the change in pathology was apparent. Conclusions The multiple low-dose streptozotocin injection protocol is a reasonable method for inducing mouse diabetic nephropathy.