Oxidative stress in diabetes coexists with a reduction in the antioxidant status, which can further increase the deleterious effects of free radicals. Berberine is one of the main alkaloids of Rhizoma coptidis which has been used to treat diabetes for more than 1400 years in China. The present study was designed to evaluate the protective effects of berberine against beta cell damage and antioxidant of pancreas in diabetic rats. Diabetic rats with hyperlipidemia were induced by intraperitoneally injection 35 mg/kg streptozotocin and a high-carbohydrate/high-fat diet. Rats were divided into 7 groups at the end of week 16: untreated control, untreated diabetic, 75, 150, 300 mg/kg berberine-treated diabetic, 100 mg/kg fenofibrate-treated, and 4 mg/kg rosiglitazone-treated. After 16 weeks treatment, serum insulin level, insulin expression in pancreas, and malonaldehyde content, superoxide dismutase activity in pancreatic homogenate were assayed. Pancreas was examined by hematoxylin/eosin staining and transmission electron microscope. Pancreas to body weight ratio, insulin level, insulin sensitivity index, malonaldehyde content and superoxide dismutase activity were altered in diabetic rats, and were near control levels treated with 150, 300 mg/kg berberine. Mitochondrial vacuolization and swelling, dilatation of the endoplasmic reticulum were observed in beta cells of diabetic rats. The pancreatic islet area atrophied and secretory granules of beta cells decreased in diabetic rats. Slight pathological changes existed in beta cells of 150, 300 mg/kg berberine-treated diabetic pancreas. These findings suggest that berberine has protective effect for diabetes through increasing insulin expression, beta cell regeneration, antioxidant enzyme activity and decreasing lipid peroxidation.