Proteolytic activity in sheared human umbilical vein endothelial cells (HUVECs) was measured utilizing a fluorogenic substrate and laser scanning confocal microscopy to clarify the key role of an intracellular Ca²⁺-sensitive protease, calpain, in these cells in response to shear stress. Within physiological shear range, activity in the cells was enhanced in shear-dependent fashion. Short interference RNA-induced silencing of m-calpain, but not of µ-calpain, suppressed the activity. Either removal of extracellular Ca²⁺ or application of an intracellular Ca²⁺ chelator (BAPTA/AM) or non-selective cation channel blocker (Gd³⁺) reduced proteolytic activity. Furthermore, activity was suppressed by phosphatidylinositol bisphosphate (PIP₂) chelator (neomycin) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002); in contrast, activity, which was partially inhibited by ERK kinase inhibitor (U0126, PD98059), was unaffected by PLC inhibitor (U73122). Moreover, Akt phosphorylation downstream of PI3K, which was elicited by shear, was attenuated by neomycin but not by calpain inhibitor (calpeptin). Following assessment of shear stress-induced focal adhesion (FA) and cytoskeletal dynamics employing interference reflection/green fluorescence protein-actin microscopy, we found that either calpain or PI3K inhibition impaired shear stress-induced polarization of FAs via stabilization of FA structures. Additionally, HUVEC alignment and cytoskeletal remodeling, which was accompanied by calpain-mediated cleavage of vinculin and talin, were also elicited by prolonged application of shear and impaired by m-calpain knockdown. Thus, these results revealed that physiological shear stress elicits Ca²⁺ influx-sensitive activation of m-calpain in HUVECs. This activity is facilitated primarily through the PI3K pathway; furthermore, it is essential for subsequent FA reorganization and cell alignment under shear conditions.