Reactive oxygen species (ROS) are important signaling molecules. They can damage in higher dosages cellular components of the vessel wall. Main sources of ROS are NADPH oxidase (NOX) complexes. We could recently show that NOX4 is the main NOX isoform in endothelial cells. However, the regulation of NOX4 in response to different levels of shear stress in endothelial cells remains unclear. In this study, long-term (24 h) exposure of endothelial cells (HUVEC) using a plate-cone viscometer to arterial laminar shear stress (30 dyne/cm²) was leading to a significant downregulation of NOX4 (0.73±0.14 of control, n=5). In addition, laminar shear stress induced elongation of endothelial cells in the direction of flow. This reflects the endothelial phenotype in large blood vessels in vivo. Lentiviral downregulation of NOX4 (0.55±0.05 of shControl, n=4) using short hairpin RNA (sh) inhibits this elongation of cell shape in response to flow. No alterations in cell viability or cell proliferation could be detected using CellTiter-Glo Assay in different cell passages after transduction with shNOX4. However, we found an impaired ability to attach to the surface of the cell culture dish in cells with pronounced NOX4 downregulation (0.27±0.08 of shControl, n=3). These data suggest, that only cells with moderate downregulation of NOX4 remain attached. In contrast to NOX4, transcription factor NRF2 was significantly upregulated by long-term exposure of endothelial cells to arterial laminar shear stress (HUVEC, 30 dyne/cm², 24 h, 1.50±0.21 of control, n=5). Transduction with shNRF2 was leading to increased cell elongation, compared to untransduced cells or to cells transduced with shControl. This seemed to be controversial to the significant induction of NRF2 in untransduced elongated cells. Additional preliminary data support a NOX4-dependent induction of NRF2 by laminar flow. Transduction of HUVEC with shNOX4 inhibits the flow-dependent regulation of NRF2. In addition, transduction with shNRF2 leads to a slight induction of NOX4 in HUVEC (1.30±0.18 of shControl, n=9) and in the endothelial cell line HMEC-1 (1.37±0.16 of control, n=3). Finally, we cloned 5'-regulatory sequences of human NOX4 promoter containing 1251 bp upstream from the transcription start site (ATG) and the first 239 bp of exon 1. We could show with the Dual-Luciferase Reporter Assay a high basal activity of the cloned NOX4 promoter construct in well-transfectable endothelial cells (HMEC-1, 4.54±0.6 of control, n=6). Transduction with shNRF2 increased NOX4 promoter activity significantly (1.77±0.04 of control, n=6). In conclusion, these data improve the knowledge about the molecular mechanisms of NOX4 regulation in response to arterial laminar shear stress in endothelial cells. Our findings support a novel feedback mechanism involving the major endothelial NADPH oxidase isoform NOX4 and the transcription factor NRF2.