Angiogenesis is a complex process involving endothelial cell migration, proliferation, and differentiation as well as tube formation. These processes are stimulated by a variety of growth factors such as vascular endothelial growth factor (VEGF). VEGF‐induced cytoskeletal reorganization plays a crucial role in the angiogenic processes. In the present study, we evaluated the role of calpain in VEGF‐induced angiogenesis in vitro and in vivo. Human pulmonary micro‐vascular endothelial cells (PMEC) were incubated with VEGF (10–60 ng/ml) for 2–24 h, after which we measured calpain activities, protein contents of the calpain subunits and of calpastatin, endothelial mono‐layer wound repair, tube formation, and actin cytoskeleton changes. Incubation of PMEC with VEGF resulted in dose‐ and time‐dependent increases in calpain activity and protein content of calpain‐2. VEGF did not change the protein contents of calpain‐1 and the small subunit or of calpastatin. Incubation of PMEC with a VEGF receptor blocker prevented the VEGF‐induced increase in calpain activity. Inhibition of calpain activity by siRNA directed against calpain‐2 and by overexpression of calpastatin prevented VEGF‐induced increases in actin stress fibers in endothelial cells and angiogenesis. Overexpression of calpastatin also inhibits vessel formation in subcutaneous (s.c.) matrigel plugs in mice. These results indicate that calpain mediates VEGF‐induced angiogenic effects by modulating actin cytoskeletal organization.—Su, Y., Cui, Z., Li, Z., Block E. R. Calpain‐2 regulation of VEGF‐mediated angiogenesis. FASEB J. 20, 1443–1451 (2006)