AbstractObesity and type‐II diabetes are growing major health issues worldwide. They are the leading risk factors for vascular insulin resistance, which plays an important role in the pathogenesis of cardiovascular disease, the leading cause of death in developed nations. Recent studies have shown that reduced synthesis of nitric oxide (NO; a major vasodilator) from L‐arginine in endothelial cells is a major factor contributing to the impaired action of insulin in the vasculature of obese and diabetic subjects. The decreased NO generation results from a deficiency of (6R)‐5,6,7,8‐tetrahydrobiopterin [BH4; an essential cofactor for NO synthase (NOS)], as well as increased generation of glucosamine (an inhibitor of the pentose cycle for the production of NADPH, another cofactor for NOS) from glucose and L‐glutamine. Accordingly, endothelial dysfunction can be prevented by (1) enhancement of BH4 synthesis through supplementation of its precursor (sepiapterin) via the salvage pathway; (2) transfer of the gene for GTP cyclohydrolase‐I (the first and key regulatory enzyme for de novo synthesis of BH4); or (3) dietary supplementation of L‐arginine (which stimulates GTP cyclohydrolase‐I expression and inhibits hexosamine production). Modulation of the arginine–NO pathway by BH4 and arginine is beneficial for ameliorating vascular insulin resistance in obesity and diabetes. © 2009 International Union of Biochemistry and Molecular Biology, Inc.