Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD1 and 3 expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD1 and 3.