Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. Endothelial dysfunction is a major factor contributing to morbidity and mortality in diabetes mellitus. The objective of this work was to study the effect of exogenous L-Arginine supplementation on tetrahydrobiopterin (BH4) availability for nitric oxide (NO) generation in normal or diabetic rats and to clarify the role of increases synthesis of NO, a major regulator of glucose utilization by skeletal muscle, in improving glucose homeostasis in diabetes. The results showed that groups received L-Arginine has a significant increase in insulin, HDL, NO, total biopterin and BH4 while it showed significant decrease in glucose, insulin resistance, LDL, cholesterol, triglyceride, arginase, urea and ADMA. In conclusion, oral administration of L-Arginine markedly increased BH4 availability and NO. L-Arginine may be a novel nutrient, which has important implications for the prevention and treatment of diabetic patients.