Objective To determine the protective effect of conditioned medium from mesenchymal stem cells (MSCs) on the diabetic nephropathy (DN) rats after intraperitoneal osmotic mini-pump implantation. Methods Thirty Sprasue-Dawley (SD) male rats were randomly divided into 3 groups, these are, normal control group, DN model group and MSCs conditioned medium group, with 10 rats in each group. The rats of the normal control group were fed common food, while those in DN group and MSCs group were fed a high-fat high-sucrose diet. The MSCs group was implanted with Alzet osmotic pump after DN models were confirmed. Blood glucose, serum insulin, triglycerides and other indices were tested in the 3 groups of rats. Renal structural damage was evaluated by periodic acid-Schiff (PAS) staining. Expression of nephrin in the renal tissues was detected by Western blotting. Results The blood glucose, serum insulin, 24-hour urinary protein, serum creatinine and blood urea nitrogen were significantly higher in the MSCs group than in the DN group (P<0.05), and the glomerular mesangial area and the glomerular volume were obviously decreased (P<0.05). The MSCs group had significantly decreased expression of nephrin protein than the normal control group (P<0.05), but the level was higher in comparison with that of the DN group (P<0.05). Conclusion Continuous pumping of conditioned medium from mesenchymal stem cells into abdominal cavity exerts a protective effect on the renal function of DN rats.