Proliferation and migration of smooth muscle cells (SMC) require myosin II activity; thus, we examined whether blebbistatin, a cell-permeable selective inhibitor of myosin II ATP activity, would impair neointimal hyperplasia after vascular injury. Delivery of blebbistatin via a perivascular polymer cuff reduced neointimal formation by 73% and luminal obstruction by 75% after carotid denudation injury in C57BL/6 mice. Blebbistatin treatment was also associated with a reduction in cell density within the neointima; total number of cells (76 ± 7 to 27 ± 3 cells/high-powered field) and actin-positive cells (64 ± 4 to 24 ± 2 cells/high-powered field) in the neointima were reduced in blebbistatin-treated mice compared with vehicle-treated mice. In a model of vascular injury with an intact endothelium, implantation of a blebbistatin-secreting cuff after carotid ligation in FVB/N mice was associated with a 61% decrease in neointimal area and a significant decrease in luminal obstruction (88 ± 4% in vehicle-treated mice versus 36 ± 4% in blebbistatin-treated mice; p < 0.0001). In cultured rat aortic SMC, blebbistatin disrupted cellular morphology and actin cytoskeleton structure, and these effects were rapid and completely reversible. Blebbistatin had a dose-dependent inhibitory effect on DNA replication and cell proliferative responses to platelet-derived growth factor-BB, angiotensin II, and α-thrombin, migratory responses to serum, and migratory responses after blunt injury. In summary, perivascular delivery of blebbistatin reduced neointimal hyperplasia after carotid injury in the mouse.