Aim To explore the possibility of endothelial nitric oxide synthase (eNOS)gene transfer preventing restenosis of vein grafts. Methods We constructed the recombinant adenovirus vector that coding eNOS, AdCMV eNOS and adenovirus vector (AdCMV). Artery bypass animal model was constructed: we took jugulars of goat as grafts, infected AdCMV eNOS and AdCMV in vitro, then anastomosed the vein grafts between carotids of goats. The functional expression of eNOS in vein grafts was assessed using immunohistochemical staining and measurement of NO concentration. The inhibition of intimal hyperplasia in vein grafts transducted AdCMV eNOS was assessed using assay of 3 H-TDR incorporation, histologic analysis, measursment of intimal thickness and area percentage of stenosis of vein grafts. Results Expression of eNOS gene in vein grafts began on 48 h after transducted with AdCMV eNOS. Levels of NO in vein grafts: AdCMV eNOS transduced veins yielded 1724.1±312.2 μmol/(L·g ), whereas AdCMV yielded 245.8±17.4 μmol/(L·g) (p<0.001, n=3). Levels of 3 H-TDR Incorporation: Incorporation in AdCMV veins was 487.7±51.1 cmp/mg vessel, and 140.8±32.5 cmp/mg vessel in AdCMV eNOS veins (p<0.01, n=3). Mean thickness of intimal was 125.7±30.60 μm in AdCMV veins, and 28.8±5.24 μm in AdCMV eNOS veins; area percentage of stenosis was 52.18%±8.46% in AdCMV veins, and 23.6%±4.71% in AdCMV eNOS veins. Conclusions Adenovirus-mediated eNOS gene transfer could inhibit intimal hyperlasia effectively. This work lay a foundation in gene therapy of grafts restenosis after CABG.