A chemokine-like factor 1 (CKLF1) is a recently discovered chemokine with broad-spectrum biological functions in inflammation and autoimmune diseases. C19 as a CKLF1's C-terminal peptide has been reported to exert inhibitory effects in a variety of diseases. However, the roles of CKLF1 and C19 on vascular smooth muscle cell (VSMC) migration and neointima formation still remain elusive. The effects of CKLF1 and C19 on VSMC migration and neointimal formation were investigated in cultured VSMCs and balloon-injured rat carotid arteries based on techniques including adenovirus-induced CKLF1 overexpression, gel based perivascular administration of C19, Boyden chamber, scratch-wound assay, real-time PCR, western blot and immunohistochemical analysis. CKLF1 was noticed to accumulate preferentially in neointima after the injury and colocalize with VSMCs. Luminal delivery of CKLF1 adenovirus to arteries exacerbated intimal thickening while perivascular administration of C19 to injured arteries attenuated this problem. In cultured primary VSMCs, CKLF1 overexpression up-regulated VSMC migration, which was down-regulated by C19. These data suggest that CKLF1 has a pivotal role in intimal hyperplasia by mediating VSMC migration. C19 was demonstrated to inhibit CKLF1-mediatated chemotaxis and restenosis. Thus further studies on C19 may provide a new treatment perspective for atherosclerosis and post-angioplasty restenosis.