Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelial dysfunction (ED) in salt-induced hypertension. The obese Zucker rat (ZR) is a model of type 2 diabetes showing hypertension and vascular ED. This study tests the hypothesis that HO-derived CO contributes to arteriolar ED and hypertension in obese ZR. Male obese (548 ± 12 g, n = 22) and lean (309 ± 14 g, n = 12) ZR were used for the study. Obese ZR had elevated mean arterial blood pressure (154 ± 3 mm Hg vs. 121 ± 4 mm Hg), blood glucose (186 ± 7 mg/dL vs. 140 ± 3 mg/dL) and blood carboxyhemoglobin (HbCO) levels (3.9 ± 0.1% vs. 3.0 ± 0.1%). Experiments used isolated skeletal muscle arterioles with constant (80 mm Hg) pressure and no flow, or constant midpoint, but altered endpoint pressures to establish graded levels of luminal flow (0-50 μL/min). In obese ZR arterioles, responses to an endothelium-dependent vasodilator, acetylcholine (ACh, 1 nmol/L - 3 μmol/L) (Δmax 32 ± 4 μm, n = 8 vs. 62 ± 7 μm, n = 5) and flow (Δmax-1 ± 1 μm, n = 4 vs. 21 ± 2 μm, n = 4) were attenuated. Acute in vitro pretreatment with a HO inhibitor, chromium mesoporphyrin (CrMP, 15 μmol/L), enhanced ACh (Δmax 59 ± 8 μm, n = 7 vs. 58 ± 13 μm, n = 5) and flow-induced dilation (Δmax 19 ± 2 μm, n = 4 vs. 20 ± 3 μm, n = 3) and abolished the differences between lean and obese ZR arterioles. Furthermore, exogenous CO (100 μmol/L) prevented the restoration of flow-induced dilation by the HO inhibitor (Δmax-1 ± 3 μm, n = 4) in obese ZR arterioles. In awake obese ZR instrumented with chronic femoral arterial catheters, administration of a HO inhibitor (25 μmol/kg/day zinc deuteroporphyrin 2,4-bisglycol IP for 3 days) lowered the blood pressure (151 ± 1 to 109 ± 1 mm Hg, n = 3). These data show that HbCO levels are increased, and arteriolar endothelium-dependent vasodilation is decreased in obese ZR. Acute in vitro treatment with a HO inhibitor restores endothelium-dependent responses to lean ZR levels, but exogenous CO prevents this effect. Furthermore, HO inhibition lowers blood pressure in obese ZR. These results suggest that heme-derived CO production is increased and contributes to arteriolar ED and hypertension in obese ZR, and hence identify vascular HO as a novel therapeutic target to prevent ED and hypertension in obesity and type 2 diabetes.