Oxidative stress is a key pathomechanism of cardiovascular diseases. However, attempts to exploit this with antioxidants have failed in clinical trials. Thus, alternative approaches are needed, i.e. to inhibit the relevant sources of reactive oxygen species (ROS). Here we aimed to establish the role of NADPH oxidases (NOX) as sources of ROS in hypertension and cerebral ischemia/reperfusion (I/R) injury. Aortas from aged spontaneously hypertensive rats (SHR) and WKY rats were used to determine ROS levels, NOX expression and localisation, as well as endothelial function. Ischemic strokes were induced by transient middle cerebral artery occlusion and photothrombosis in wildtype and NOX4 knockout mice of both sexes and different ages. NOX expression, infarct areas, oxidative stress markers, blood-brain barrier breakdown, apoptosis, neurological scores and survival were assessed. Aged SHR displayed endothelial dysfunction and increased vascular ROS levels compared to aged-matched WKY rats. Inhibition of NADPH oxidases with VAS2870 normalised aortic ROS levels and restored endothelial function in SHR aortas. The NADPH oxidase isoforms NOX1 and NOX2 were upregulated in SHR aortas with NOX1 displaying ectopic expression in the endothelium. In contrast, NOX4 levels were unchanged, and NOX4 knockout mice displayed normal blood pressures. A major pathophysiological role for NOX4 became evident in I/R induced tissue damage: NOX4 was upregulated after I/R in human and mouse brains. After acute and chronic ischemia, brains from NOX4 knockout mice were protected from oxidative stress, blood-brain barrier breakdown and neuronal apoptosis. VAS2870 application within a clinically relevant time frame, i.e. post-stroke, protected wild-type mice from stroke-induced I/R damage. NOX1 and NOX4 appear to represent promising targets to treat oxidative stress-associated hypertension and prevent I/R injury, respectively. Pharmacological inhibition of NADPH oxidases using specific compounds may pave new avenues for the treatment of hypertension and ischemic brain injury in humans.