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Abstract The human brain microvasculature is constantly exposed to variable fluid flow regimes and their influence on the endothelium depends in part on the synchronous cooperative behavior between cell–cell junctions and the cytoskeleton. In this study, we exposed human cerebral microvascular endothelial cells to a low laminar flow (1 dyne⋅cm⁻²), high laminar flow (10 dyne⋅cm⁻²), low oscillatory flow (±1 dyne⋅cm⁻²), or high oscillatory flow (±10 dyne⋅cm⁻²) for 24 hr. After this time, endothelial cell–cell junction and cytoskeletal structural response was characterized through observation of zonula occludens‐1 (ZO‐1), claudin‐5, junctional adhesion molecule‐A (JAM‐A), vascular endothelial cadherin (VE‐Cad), and F‐actin. In addition, we also characterized cell morphology through measurement of cell area and cell eccentricity. Our results revealed the greatest change in junctional structure reorganization for ZO‐1 and JAM‐A to be observed under low laminar flow conditions while claudin‐5 exhibited the greatest change in structural reorganization under both low and high laminar flow conditions. However, VE‐Cad displayed the greatest structural response under a high laminar flow, reflecting the unique responses each cell–cell junction protein had to each fluid flow regime. In addition, cell area and cell eccentricity displayed most significant changes under the high laminar flow and low oscillatory flow, respectively. We believe this study will be useful to the field of cell mechanics and mechanobiology.