Oxidative stress is considered as cardiovascular risk factor. In the vessel wall, nicotine adenine dinucleotide phosphate (NADPH) oxidase complexes have been identified as main source of oxidative stress. Expression of novel NADPH oxidase isoformes in endothelial cells is not well understood. Endothelial cells are constantly exposed to shear stress by flowing blood. Recent evidence indicates a regulation of NADPH oxidase isoformes by flow. Here we show that NADPH oxidase subunit Nox4 is the major Nox isoform in human endothelial cells. Nox4 is localized in the perinuclear space and enhances endothelial superoxide anion formation by overexpression. Long-term application of arterial laminar shear stress was downregulating superoxide anion formation and Nox4 expression. Molecular cloning and functional analysis of the human Nox4 promoter revealed that an AP-1 binding site is essential for downregulation of Nox4 by laminar shear stress. In addition, downregulation of DNA binding activity of a c-jun-containing AP-1 transcription factor in response to long- term laminar shear stress was observed. This novel mechanism could be involved in the vasoprotective downregulation of endothelial oxidative stress in response to shear stress.