Application of shear stress on endothelial cells induced a conversion from a venous to an arterial phenotype, including elongation and alignment of the cells. Recently we showed that rac1, a small GTPase of the rho family and an activator for endothelial NADPH-Oxidase, is essential to mediate shear stress induced cell alignment, migration and elongation. Here we show that the antioxidant idebenone completely inhibits the shear stress (12 dyn/cm2) induced cell elongation and alignment of the cells. The same effect could be obtained when NADPH-Oxidase, an important source for reactive oxygen species (ROS), was inhibited by diphenyliodonium chloride. To obtain direct evidence for the role of NADPH-oxidase we constructed a lentiviral shRNA against Nox4, a subunit of the NADPH-oxidase isoform in endothelial cells. Down-regulation of Nox4 also largely blocked the elongation and alignment of the cells. Together this data show that shear stress induced rac1 activation results in a NADPH-Oxidase dependent ROS-Production, which is a critical signalling event for cell elongation and alignment under flow conditions.