Aims Endothelial dysfunction is an early step in the development of atherosclerosis. Increased formation of superoxide anions by NADPH oxidase Nox1, 2, and 5 reduces nitric oxide availability and can promote endothelial dysfunction. In contrast, recent evidence supports a vasoprotective role of H₂O₂ produced by main endothelial isoform Nox4. Therefore, we analysed the impact of genetic deletion of Nox4 on endothelial dysfunction and atherosclerosis in the low-density lipoprotein receptor (Ldlr) knockout model. Methods and results Ex vivo analysis of endothelial function by Mulvany myograph showed impaired endothelial function in thoracic aorta of Nox4^-/-/Ldlr^-/- mice. Further progression of endothelial dysfunction due to high-fat diet increased atherosclerotic plaque burden and galectin-3 staining in Nox4^-/-/Ldlr^-/- mice compared with Ldlr^-/- mice. Under physiological conditions, loss of Nox4 does not influence aortic vascular function. In this setting, loss of Nox4-derived H₂O₂ production could be partially compensated for by nNOS upregulation. Using an innovative optical coherence tomography approach, we were able to analyse endothelial function by flow-mediated vasodilation in the murine saphenous artery in vivo. This new approach revealed an altered flow-mediated dilation in Nox4^-/- mice, indicating a role for Nox4 under physiological conditions in peripheral arteries in vivo. Conclusions Nox4 plays an important role in maintaining endothelial function under physiological and pathological conditions. Loss of Nox4-derived H₂O₂ could be partially compensated for by nNOS upregulation, but severe endothelial dysfunction is not reversible. This leads to increased atherosclerosis under atherosclerotic prone conditions.