Tetrahydrobiopterin (BH₄) is an essential cofactor of nitric oxide synthase that improves endothelial function in diabetics, smokers and in patients with hypercholesterolemia. Insulin resistance has been suggested a contributing factor in the development of endothelial dysfunction via an abnormal pteridine metabolism. We hypothesized that BH₄ would restore flow-mediated vasodilation [FMD] (endothelial-dependent vasodilation), which may affect insulin resistance in type 2 diabetic patients. Thirty-two subjects (twelve type 2 diabetic subjects, ten matched non-diabetic subjects and ten healthy unmatched subjects) underwent infusion of BH₄ or saline in a random cross-over study. Insulin sensitivity index (S_I) was measured by hyperinsulinemic isoglycemic clamp. FMD was measured using ultrasonography. BH₄ significantly increased S_I in the type 2 diabetics (3.6±0.6 vs. 4.9±0.7 10^-4 dl^.kg^-1.min^-1/(µU/mL), P<0.05), while having no effects in non-diabetics (8.9±1.1 vs. 9.0±0.9 10^-4 dl^.kg^-1.min^-1/(µU/mL), P=0.92) or in healthy subjects (17.5±1.6 vs. 18±1.8 10^-4 dl^.kg^-1.min^-1/(µU/mL), P=0.87). BH₄ did not affect the relative changes in brachial artery diameter from baseline FMD(%) in type 2 diabetic subjects (2.3±0.8 vs. 1.8±1.0 %, P=0.42), non-diabetic subjects (5.3±1.1 vs. 6.6±0.9 %, P=0.32) or in healthy subjects (11.9±0.6 vs. 11.0±1.0 %, P=0.48). In conclusion, BH₄ significantly increases insulin sensitivity in type 2 diabetic patients without any discernable improvement in endothelial function.