Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complimentary techniques photoactivation of fluorescence and fluorescence recovery after photobleaching, we characterize endothelial actin cytoskeleton dynamics during the alignment process in response to steady, laminar fluid flow and correlate these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movements by 50%. Although movement slows, the actin filament turnover rate increases 3-fold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by disperse, non-confluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells where cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returns to pre-shear stress levels, but actin remodeling remains highly dynamic in many cells following alignment suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin remodeling response that is used for endothelial cell shape change instead of bulk cell translocation.