Abstract Hemodynamic forces play an important role in vascular network development and homeostasis. In physiological condition, shear stress generated by laminar flow promotes endothelial cells (EC) health and induces their alignment in the direction of flow. In contrast, altered hemodynamic forces induce endothelial dysfunction and lead to the development of vascular disorders such as atherosclerosis and aneurysms. Following mechano-sensor activation, Rho protein-mediated cytoskeletal rearrangement is one of the first steps in transforming flow-induced forces into intracellular signals in EC via guanine nucleotide exchange factors (RhoGEFs) that mediate the spatio-temporal activation of these Rho proteins. Here we identified ARHGEF18 as a flow-sensitive RhoGEF specifically activating RhoA. Both ARHGEF18 expression and activity were controlled by shear stress level. ARHGEF18 promotes EC adhesion, focal adhesion formation and migration. ARHGEF18 localized to the tight junction by interacting with ZO-1 and participated to shear stress-induced EC elongation and alignment via its nucleotide exchange activity and the activation of p38 MAPK. Our study therefore characterized ARHGEF18 as the first flow-sensitive RhoA GEF in ECs, whose activity is essential for the maintenance of intercellular junctions and a properly organized endothelial monolayer under physiological flow conditions.