Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 hours post fertilization during zebrafish (Danio rerio) development. Forty eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1 to 4-cell stage, zebrafish demonstrated various cardiovascular defects including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the [Tg(fli1:EGFP)y1] zebrafish line (in which a fli1 promoter drives vascular specific EGFP expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was downregulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibitor of this pathway blocked calponin 2’s promigratory effect. Collectively, these data suggest that calponin 2 plays an important role in migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.