Ageing is an independent risk factor of cardiovascular diseases which involves oxidative stress derived from a Nox2-containing NADPH oxidase. However, the mechanism of endothelial Nox2 activation in normal ageing process remains unclear. In this study, we investigated the therapeutic potential of targeting Nox2 in improving global metabolism and endothelial function at old age by using age-matched wild-type and Nox2 knockout mice at 3–4 months (young); 11–12 months (middle aged) and 21–22 months (ageing). Compared to young mice, middle-aged and ageing wild-type mice had significantly higher blood pressure, hyperglycaemia, hyperinsulinaemia. These were accompanied by oxidative stress in multiple organs including the lung, the liver, the heart and vessels. The vessel motor function was examined in an organ bath using aortas isolated from these mice. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in aortas of wild-type ageing mice, and this was accompanied by increased expressions of Nox2 and markers of inflammation, activation of MAPK and Akt and decreased insulin receptor expression and function. However, these aging-associated disorders in the aortas were significantly reduced by knocking out Nox2 in mice. In response to high glucose plus high insulin challenge, coronary microvascular endothelial cells isolated from wild-type mice displayed significantly increased Nox2 expression, oxidative stress and cell senescence, e.g. increase p53 expression and β²–galactosidase activity. However, these responses were absent or significantly reduced in endothelial cells isolated from Nox2 knockout mice. In conclusion, metabolic disorders in particular hyperglycaemia and insulin resistance play an important role in mediating Nox2 activation and oxidative stress in multiple organs in ageing. Nox2 is involved in normal ageing process-associated vascular inflammation and oxidative damage of endothelial dysfunction.