Endothelial cells are known to adapt their morphology and functions in response to different types of fluid flow. In vivo, endothelial cells are exposed to varying frequencies of pulsatile flow. Therefore, in this study, the effects of frequency of pulsatile flow on morphology and alpha v beta 3 integrin expression of bovine aortic endothelial cells (BAEC) were investigated. A newly developed flow-system was used to apply pulsatile flow at frequencies of 0.5, 1.0, and 1.5 Hz to BAEC for 12 h. At 1.0 Hz, the expression of alpha v beta 3 integrin was significantly higher at the central regions of cells than at the upstream regions. In contrast, at 0.5 and 1.5 Hz, the expression of alpha v beta 3 integrin was significantly higher at the upstream regions than at the central regions. Morphological observation revealed that BAEC under pulsatile flow frequencies of 0.5 and 1.0 Hz exhibited elongated morphologies and oriented with the direction of flow. In addition, stress fibers developed parallel to the cell major axis. Similar results were obtained under steady flow conditions. In contrast, BAEC under 1.5 Hz elongated but with a random orientation. These results suggest that BAEC morphology and integrin expression may be modulated by the frequency of pulsatile flow. In particular, BAEC exposed to 1.5 Hz pulsatile flow may not be able to align with the direction of flow, possibly indicating the presence of a critical value for living systems.