Diabetes mellitus is characterized by impaired endothelial cell production of the vasodilator and anti platelet adhesion factor nitric oxide (NO) .This study aimed to evaluate the role of exogenous L-arginine in diabetic rats in context of regulation of Asymmetrical Dimethylarginine (ADMA) and NO levels. Seventy five male albino rats were used in this study and divided into five groups ; Group I (Control group): healthy rats received a vehicle , Group II (L-arginine group): healthy rats received L-arginine (10 mM /Kg b.w./day ) orally for eight weeks, Group III (Diabetic group): diabetic rats received a vehicle, Group IV (Treated group): diabetic rats received L-arginine (10 mM /Kg b.w./day )orally for eight weeks, Group V (Prophylactic group): healthy rats received L-arginine (10 mM /Kg b.w./day ) orally before( two weeks ) and after ( eight weeks) induction of diabetes. Fasting blood sugar, insulin, blood urea, serum arginase and nitric oxide were determined. ADMA was determined by HPLC; separation was achieved on reversed phase column (150 X 4.6 mm C18).Mobile phase was eluted by gradient method; flow-rate was 1.0 ml/min. The wavelengths of fluorescence detector were set at 338 and 425 nm. In diabetic rats, serum ADMA was significantly increased along with the reduction of NO; however, L-arginine supplementation significantly decreased serum ADMA and increased NO in both prophylactic and treated groups. In conclusion, supplementation of L-arginine is a potentially novel mean to prevent diabetes-associated endothelial dysfunction through the reduction of asymmetrical dimethylarginine and the elevation of nitric oxide.