Objective To construct an adenovirus vector containing eNOS gene，and analyze the expression and location in endothelial progenitor cells from bone marrow. Methods Establish the vector for recombinant adenovirus: the recombinant plasmid PSUCMV-heNOS was established by inserted the heNOS gene in the vector PSUCMV. After identified for correction in sequence, it was transferred to cell 293 with the plasmid including the right arm of 5-type adenovirus by Lipofectamine 2000. EPCs from bone marrow transfected by recombinant adenovirus with eNOS gene: 18 male Wistar rats, aged 4-6w, were elected for EPCs culture. The cells in the third passages were randomly allocated to 3 groups: PBS control group (Cgroup), Lac gene transfection group(Lgroup) and the eNOS gene transfection group(N group). Every group was separated into 3 subgroups following the 1d, 3d and 7d after treatment. The changes in the morphology, the product of NO in the culture medium and the express of eNOS were monitored. Results The DNA of AdCMVheNOS was successfully transferred into the 293 cells by liposome and packaged inside. After confirmed by PCR analysis, the titer was 1.58?010PFU/ml after amplified. The culture of EPCs from bone marrow in rat was observed, and the expression of eNOS product was increased after transfected for 1 day. At the same time point, the content of eNOS in N group was significantly higher than the L group and C group. The N7 and N3 subgroup showed a significant difference compared with the N1 subgroup. The production of NO increased following the time and the difference was statistic between the 3 day and the 7 day subgroups. Conclusion Adenovirus can mediate the high quality expression of eNOS gene in the EPCs and induce the increasement of NO in culture medium and eNOS expression in cells.