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Diabetic rats exhibit decrease in plasma arginine, NO synthesis and tetrahydrobiopterin in endothelial cells (EC). Treatment with L-arginine may be beneficial for enhancing NO synthesis in diseases associated with endothelial dysfunction. However, little is known about the mechanism responsible for the stimulatory effect of arginine on endothelial NO synthesis. We hypothesized that dietary arginine supplementation increases BH4 for NO synthesis in EC of diabetic rats, thereby preventing endothelial dysfunction. In experiment I, streptozotocin (STZ) induced-diabetic male Sprague Dawley (SD) rats (a model of type-I diabetes) were individually pair-fed a casein-based diet on the basis of feed intake (per kg body weight) of non-diabetic SD rats. Addition of arginine-HCl or alanine to drinking water for the rats were adjusted daily to ensure isonitrogenous provision per kg body weight. In non-diabetic rats, arginine supplementation increased plasma arginine (144%), plasma insulin (44%), EC arginine (88%), EC BH4 (106%) and EC NO synthesis (80%), compared with alanine treatment. In diabetic rats, arginine supplementation reduced body weight loss (36%), and plasma glucose (54%), and increased plasma arginine (110%), plasma insulin (209%), EC arginine (173%), EC BH4 (128%) and EC NO synthesis (125%), compared with alanine treatment. In experiment II, male Zucker diabetic fatty (ZDF) rats (a model of type-II diabetes) were individually pair-fed a Purina 5008 diet on the basis of feed intake by alanine-treated diabetic rats (per kg body wt). Addition of arginine-HCl or alanine to drinking water for the rats was adjusted daily to ensure isonitrogenous provision per kg body weight. Arginine supplementation to ZDF rats did not affect plasma glucose and insulin, reduced epidididmal fat (30%), abdominal fat (43%) and body weight gain (18%), and increased plasma arginine (273%), EC arginine (197%), EC BH4 (120%) and EC NO synthesis (122%), compared with alanine-treated ZDF rats. These results show that dietary L-arginine supplementation increases BH4 and NO synthesis in EC of both STZ-diabetic and ZDF rats. Strikingly, arginine treatment prevented hyperglycemia in STZ-diabetic SD rats and reduced obesity in ZDF rats. Collectively, results demonstrate that oral administration of arginine is beneficial for both type-I and type-II diabetic rats.