We previously observed that adenovirus-mediated expression of C-type natriuretic peptide (CNP) markedly inhibits neointima formation after balloon injury in rat carotid arteries, suggesting that CNP has multiple effects over its modest inhibitory effect on cellular proliferation. We hypothesized that local expression of CNP might have antithrombotic and antiinflammatory effects. Balloon-injured rabbit carotid arteries were infected with an adenovirus expressing human CNP (AdCNP), human tissue factor pathway inhibitor (AdTFPI), or bacterial beta-galactosidase (AdLacZ) or infused with saline. Seven days later, shear stress-induced thrombosis was evaluated by cyclic flow variation (CFV), reflecting recurrent cycles of thrombus formation and dislodgment. CFV was observed in all AdLacZ-infected and saline-infused arteries but not in arteries infected with AdCNP or AdTFPI even in the presence of epinephrine. Injury increased the expressions of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and infiltration of macrophages. However, these effects were markedly reduced in AdCNP-treated arteries but not in AdTFPI-infected ones. In AdCNP-infected arteries, injury-induced expression of inducible NO synthase (iNOS) was enhanced, leading to increased NO generation. Interestingly, when the enhanced NO production was inhibited, neither inhibitory effect was observed, and suppression of neointima formation by CNP was canceled. Our study demonstrates that overexpression of CNP shows antithrombotic and antiinflammatory effects and reduces neointima formation mainly through enhanced NO production.