Oxidative stress attributable to the activation of a Nox2-containing NADPH oxidase is involved in the development of vascular diseases and in aging. However, the mechanism of Nox2 activation in normal aging remains unclear. In this study, we used age-matched wild-type (WT) and Nox2 knockout (KO) mice at 3–4 months (young); 11–12 months (middle-aged) and 21–22 months (aging) to investigate age-related metabolic disorders, Nox2 activation and endothelial dysfunction. Compared to young mice, middle-aged and aging WT mice had significant hyperglycaemia, hyperinsulinaemia, increased systemic oxidative stress and higher blood pressure. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in WT aging aortas, and this was accompanied by increased Nox2 and ICAM-1 expressions, MAPK activation and decreased insulin receptor expression and signaling. However, these aging-associated disorders were significantly reduced or absent in Nox2KO aging mice. The effect of metabolic disorder on Nox2 activation and endothelial dysfunction was further confirmed using high-fat diet-induced obesity and insulin resistance in middle-aged WT mice treated with apocynin (a Nox2 inhibitor). In vitro experiments showed that in response to high glucose plus high insulin challenge, WT coronary microvascular endothelial cells increased significantly the levels of Nox2 expression, activation of stress signaling pathways and the cells were senescent, e.g. increased p53 and β–galactosidase activity. However, these changes were absent in Nox2KO cells. In conclusion, Nox2 activation in response to aging-associated hyperglycaemia and hyperinsulinaemia plays a key role in the oxidative damage of vascular function. Inhibition or knockout of Nox2 preserves endothelial function and improves global metabolism in old age.