Scribble (Scrib) is a highly conserved epithelial cytoplasmic polarity protein, which also acts as a tumour suppressor. We observed that Scrib is expressed in endothelial cells, although its function in the vascular system is completely elusive. As fluid shear stress is an important stimulus for endothelial cell alignment, we hypothesized that Scrib contributes to endothelial cell mechano-transduction. Scrib localization was determined in the murine aorta and the effect of shear stress on endothelial Scrib positioning was analysed in human umbilical vein endothelial cells (HUVEC) by confocal microscopy. The involvement of Scrib in shear stress signaling was determined with Scrib siRNA. In the mouse aorta, Scrib was exclusively present in the endothelium and co-localized with VE-cadherin. In HUVEC cultured under static conditions, the protein was also mainly present in the plasma membrane. Unidirectional laminar flow as generated by the ibidi-flow system induced an alignment of the cells and a positioning of the golgi apparatus behind the nucleus in the flow direction. Shear stress also resulted in a translocation of Scrib to the leading part of the cell diametric to the flow direction. HUVEC subjected to Scrib siRNA and laminar flow showed an increased cell length and size compared to scrambled siRNA transfected cells and an enhanced flow-induced alignment. Moreover the position of the Golgi apparatus after Scrib siRNA treatment changed from back to front position relative to the nucleus. Taken together, these observations identify Scrib as a novel negative regulator of endothelial shear stress signalling. Inhibition of Scrib could be a strategy to enhance the beneficial effects of laminar flow on endothelial cell function.