Shear stress generated by flowing blood has major effects on vascular function. Bends and branches of arteries are exposed to complex blood flow patterns, a mechanical environment that promotes cardiovascular dysfunction and atherosclerosis. The exact mechanisms, by which endothelial cells translate shear stress to physiological and pathophysiological responses remain incompletely understood. Here, we identified perturbations of the ubiquin landscape in endothelial cells driven by shear stress. The integrative analysis of proteome and ubiquitome revealed that non-degradative ubiquitination controls cytoskeleton rearrangements in response to shear stress. These results provide a global view of the contribution of the ubiquitin system during shear stress response.