Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10Pa with SSG of up to 34Pa/mm for 24 and 72h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.