Background: Diabetic nephropathy (DN) significantly contributes to the mortality rates among diabetic individuals. A robust animal model is crucial for DN and related research. However, current models present various limitations. Methods: Mus musculus castaneus mice were administered a high-fat diet for 4 weeks, followed by an injection of streptozotocin (STZ) at a dosage of 40 mg/kg/day for 5 consecutive days, i.p.. After a 7-day period, fasting blood glucose (FBG) levels were measured to determine the onset of diabetes. These diabetic mice continued access to the high-fat diet until the development of DN, which was confirmed by urea nitrogen (BUN), serum creatinine (Scr), urinary albumin, and renal pathological observation. Serum insulin was measured by ELISA method. Results: The model group exhibited persistently high levels of FBG, accompanied by a significant reduction in body weight. Following an additional 4 weeks on the high-energy diet, the diabetic mice displayed a decline in insulin level, whereas BUN, Scr, urinary albumin, kidney index, and glomerular volume all demonstrated significantly elevations. Histological examinations revealed progressive mesangial matrix expansion, partial fused foot process, and thickening glomerular basement membrane. In contrast, high-diet control mice exhibited mild renal impairment, yet their FBG and insulin levels were comparable to those of the normal mice. Conclusion: A robust model of DN was established in Mus musculus castaneus mice within 4 weeks after confirmation of diabetes, which was induced by a combination of feeding a high-fat diet for 4 weeks and repeated exposure to low-dose STZ. Notably, a high-fat diet alone did not induce DN under the experimental conditions.