Objective:To establish an effective method to transfect canine bone marrow-derived EPCs with human eNOS gene(heNOS). Methods:Canine endothelial progenitor cells(EPCs)were obtained,cultured and expanded with bone marrow-derived mononeuclear cells by our previously published ex vivo expansion method,then transfected by Ad5-heNOS recombinant adenovirus or pEGFP-N1-heNOS recombinant plasmid,EPCs without transfection as a control. The expression of heNOS protein in EPCs was measured by enzyme linked immunosorbent assay(ELISA) at 48 hours after gene transfection. The amount of the nitric oxide(NO) in the supernanant of culture fluid was detected by nitrate reductase assay. Results:The expression of heNOS protein in Ad5-heNOS transfected EPCs(2 091.67 ± 172.49 pg/ml) was significantly higher than that in pEGFP-N1-heNOS transfected EPCs(173.67 ± 36.76 pg/ml) and the control group (158.00 ± 30.91 pg/ml)(P < 0.01). The amount of NO in the supernatant of the Ad5-heNOS group(49.5 ± 5.2 μmol/L) was significantly higher than that in the pEGFP- N1-heNOS group(38.5 ± 7.1 μmol/L) and the control group(39.7 ± 7.2 μmol/L). Conclusions:The heNOS gene can be successfully transfected into and expressed in canine EPCs by Ad5-heNOS recombinant adenovirus.