Endothelial cells (ECs) sense shear stress and transduce blood flow information into functional responses that play important roles in vascular homeostasis and pathophysiology. A unique feature of shear-stress-sensing is the involvement of many different types of membrane-bound molecules, including receptors, ion channels, and adhesion proteins, but the mechanisms remain unknown. Because cell membrane properties affect the activities of membrane-bound proteins, shear stress may activate various membrane-bound molecules by altering the physical properties of EC membranes. To determine how shear stress influences the cell membrane, cultured ECs were exposed to shear stress and examined for changes in membrane lipid order and fluidity by Laurdan two-photon imaging and FRAP measurements. Upon shear stress stimulation, the lipid order of EC membranes rapidly decreased in an intensity-dependent manner, and caveolar membrane domains changed from the liquid-ordered state to the liquid-disordered state. Notably, a similar decrease in lipid order occurred when the artificial membranes of giant unilamellar vesicles were exposed to shear stress, suggesting that this is a physical phenomenon. Membrane fluidity increased over the entire EC membranes in response to shear stress. Addition of cholesterol to ECs abolished the effects of shear stress on membrane lipid order and fluidity and markedly suppressed ATP release, which is a well-known EC response to shear stress and is involved in shear-stress Ca2+ signaling. These findings indicate that EC membranes directly respond to shear stress by rapidly decreasing their lipid phase order and increasing their fluidity; these changes may be linked to shear-stress-sensing and response mechanisms.