Hyperleptinemia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide (O₂^-), and peroxynitrite (ONOO^-) nanosensors were placed near the surface (5 ± 2µm) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin, or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI)-stimulated NO, O₂^-, ONOO^- were recorded. Endothelial nitric oxide synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did not directly stimulate NO, O₂^-, or ONOO^- release from HUVEC. However, chronic (12hr) exposure of HUVEC to leptin increased eNOS expression by 250%, and CaI-stimulated NO (625 ± 30 vs. 500 ± 24nmol/L control) and dramatically increased cytotoxic O₂^- and ONOO^- levels. The [NO]/[ONOO^-] ratio decreased from 2.2 ± 0.1 in normal to 1.30 ± 0.05 in leptin induced dysfunctional endothelium. In obese mice, 2.5-fold, increase in leptin concentration coincided with 230 ± 15% increase in eNOS and about 30% decrease in intracellular L-arginine. The increased eNOS expression and reduced L-arginine content led to eNOS uncoupling, reduction in bioavailable NO (250 ± 10 vs. 420 ± 12nmol/L control) and about 40% increase in O₂^- and ONOO^- concentration. L-arginine and sepiapterin supplementation reversed eNOS uncoupling and partially restored [NO]/[ONOO^-] balance in obese mice. In obesity, leptin increases eNOS expression and decreases intracellular L-arginine, resulting in eNOS uncoupling and depletion of endothelial NO and increase of cytotoxic ONOO^-. Hyperleptinemia triggers an endothelial [NO/ONOO^-] imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e. atherosclerosis, diabetes.