Objective: To investigate the effect of human eNOS (heNOS) gene transfection on the functions of canine bone marrow-derived endothelial progenitor cells (EPC). Methods: Canine EPCs were transfected with the recombinant adenovirus vector type 5 carrying heNOS gene (Ad5-heNOS) after directional culture and expansion ex vivo, and the untransfected canine EPCs were used as control. After transfection, the heNOS protein expression and NO production of EPCs were measured by enzyme linked immunosorbent assay (ELISA) and nitrate reductase assay, respectively. The EPCs functions including proliferation, adhesion, migration and anti-senescence as well as angiogenesis ability were also determined. Results: Forty-eight hours after transfection with Ad5-heNOS, the heNOS protein expression and NO production of transfected EPCs were significantly higher than those of the untransfected EPCs ［(2091.67±172.489) pg/mL vs. (158.00±30.914) pg/mL; (49.5±5.16) μmol/L vs. (39.7±7.24) μmol/L］ (both P＜0.01). The cell number of proliferation, adhesion and migration of the transfected EPCs were all significantly higher than those of the untransfected EPCs (0.52±0.03 vs. 0.31±0.02; 28.00±1.41 vs. 11.83±1.45; 109.67±6.95 vs. 72.67±6.29) (all P＜0.01), while the percentage of senescent cells of the transfected EPCs was significantly lower than that of the untransfected EPCs (0.22±0.02 vs. 0.32±0.01) (P＜0.01). The transfected EPCs assembled into primitive vascular tube-like structures when plated in Matrigel ex vivo. Conclusions: heNOS gene transfection can promote the proliferative ability of canine EPC and also enhance its functions.