Aim To investigate the effect, safety and mechanism of adenovirus-mediated local human tissue factor pathway inhibitor (TFPI) gene on in-stent restenosis. Methods The ilio-femoral arteries of thirty six New Zealand white rabbits were inflated by balloon angioplasty catheter and implanted nickel titanium alloy self-expended stents. Adenovirus-mediated TFPI gene (Ad-TFPI), bacterial β-galactosidase gene (Ad-LacZ) and saline were irrigated for 20 minutes at the site of stents respectively. At the day of 3, 7 and 28 after injuried, experimental arteries were harvested and studied the targets such as the exogenous gene transfer rate and expression were studied, and immunohistochemistry staining (proliferating cell nuclear antigen,PCNA), TUNEL assay (terminal dUTP nick-end labeling to detect apoptotic cells), histomorphometry and blood biochemical detection were used. Results Genes were transferred and expressed successfully. Compared with Ad-LacZ and saline groups, Ad-TFPI group's vascular smooth muscle cell (VSMC) proliferation rate was significantly decreased (P<0.05 ) and VSMC apoptosis rate was significantly increased on the 7th day (P<0.05). The mean neointimal area, the ratio of the neointimal to medial areas, and percent of stenosis in the TFPI group were all significantly reduced compared with the control groups (P<0.05). The main organs and blood biochemical indicators of the treated animals were not changed in all groups. Conclusions Ad-TFPI gene transfer significantly reduced neointimal hyperplasia, suppressed cell proliferation, induced cell apoptosis without systemic side effects.