Reduction of nitric oxide (NO), a potent vasodilator, and an increase in cytotoxic peroxynitrite (ONOO⁻) may be associated with the uncoupling of NO synthase (eNOS) and endothelial cell (EC) dysfunction. In addition to its effect on glucose control, metformin, may also directly benefit in the restoration of the function of eNOS and EC. Obese Zucker rats were administered vehicle or 300 mg/kg/day metformin for 4 weeks. NO concentration [NO] and ONOO⁻ concentration [ONOO⁻] were measured in aortic and glomerular endothelial cells from Zucker rats in vitro. Compared with controls, aortic and glomerular endothelial [NO] was reduced by 32% and 41%, while [ONOO⁻] release increased 79% and 69%, respectively. Metformin treatment increased aortic and glomerular endothelial [NO] by 37% and 57%, respectively, while decreasing [ONOO⁻] by 32% and 34%, compared with vehicle-treated animals. Treatment with metformin significantly restored the balance in the [NO]/[ONOO⁻] ratio with 101% and 138% increase for aortic and glomerular endothelial cells, respectively. Fasting glucose levels were not significantly changed. These findings indicate that metformin therapy has a direct and beneficial effect on arterial and renal EC function in obese rats, including enhanced NO release and reduced nitroxidative stress, beyond any effects on fasting glucose levels.