The regulation of endothelial function plays an important role in the development and progression of metabolic and cardiovascular diseases. A critical determinant of endothelial function is the balance between nitric oxide and reactive oxygen species. Endothelium-derived NO availability can be limited by enhanced formation of reactive oxygen species. Major sources of reactive oxygen species in the vessel wall are NAD(P)H oxidase complexes. This review summarizes the impact of vascular NAD(P)H oxidase-derived reactive oxygen species on atherosclerosis and endothelial dysfunction. Changes in NAD(P)H oxidase expression and activity have clinical implications. Mutations in NAD(P)H oxidase subunits can lead to impaired oxidative burst in leukocytes and chronic granulomatous disease. In contrast, normalization of increased expression and activity of NAD(P)H oxidase in endothelial dysfunction and vascular disorders can be considered as a novel therapeutic strategy in the treatment of cardiovascular diseases.