Vascular smooth muscle cell (VSMC) migration is implicated in restenosis. Myosin binding protein H (MYBPH) is capable of reducing cell motility and metastasis. In this study, we sought to determine whether MYBPH is involved in VSMC migration and neointima formation in response to vascular injury. To determine the expression of MYBPH in injured artery, we used a standard rat carotid artery balloon-injury model. In vivo studies have demonstrated that MYBPH is upregulated after vascular injury. VSMCs treated with platelet-derived growth factor (PDGF)-BB displayed increased MYBPH mRNA and protein levels. PDGF-induced VSMC migration was inhibited by adenovirus-mediated expression of MYBPH whereas it was enhanced by small interfering RNA knockdown of MYBPH. The activation of ROCK1 was repressed by MYBPH. Luminal delivery of MYBPH adenovirus to carotid arteries decreased neointimal hyperplasia in vivo. MYBPH may, therefore, serve as a novel therapeutic target for postangioplasty restenosis.