Arterial bifurcations are known as the common sites for development of cerebral aneurysms. Although localization of aneurysms suggests that high wall shear stress (WSS) and high wall shear stress gradient (WSSG) 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 still remain unclear. In this study, we observed morphological responses of ECs under high WSS and high WSSG condition using a T-shaped flow chamber and under high WSS without WSSG condition using a parallel plate flow chamber to evaluate the effects of WSSG to ECs, After 24 h exposure to flow, ECs under high WSS (10 Pa) without WSSG condition oriented perpendicular to the flow, whereas ECs at high WSS (10 Pa) with WSSG condition did not cause EC alignment. After 72 h exposure to flow, ECs exposed to WSSG were not polarized whereas ECs at high WSS without WSSG condition orientated and elongated to the direction of flow. These results indicate that a WSSG may suppress orientation of ECs to the flow direction.