Disturbed flow leads to increased inflammatory responses of endothelial cells (ECs) prone to atherogenic state. Currently, little is known about the physiological mechanisms protecting vasculature against disturbed flow-activated ECs leading to atherosclerosis. Understanding the protective mediators involved in EC activation could provide novel therapeutic strategies for atherosclerosis. The extracellular matrix microenvironment profoundly regulates cellular homeostasis. A non-EC resident ECM protein, cartilage oligomeric matrix protein (COMP), has diverse protective roles in the cardiovascular system. To determine whether COMP could protect against disturbed flow-activated EC and atherosclerosis, we compared oscillatory shear stress (OSS) induced EC activation coated with various ECM proteins. Purified COMP inhibited EC activation caused by OSS. EC activation was upregulated in the aortic arch where the flow is disturbed in COMP−/− mice as compared with wild-type mice under physiological conditions or pathologically in partially ligated mouse carotid arteries. Mechanistically, co-immunoprecipitation, mammalian two-hybrid and FRET assay results suggest that COMP bound directly to integrin α5 via its C-terminus. We next synthesized a COMP-derived peptidomimetics (CCPep24) mimicking a specific COMP–integrin α5 interaction and found that CCPep24 protected against EC activation and atherogenesis in vivo. This study extends our current understanding of how ECM and flow coordinately fine-tune EC homeostasis and reveals the potential therapeutic effect of COMP or COMP-derived peptidomimetics on blocking aberrant integrin α5 activation, inflammatory EC activation and atherosclerosis pathogenesis.