Background and Objective: Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS), which is expressed widely in tumor tissue, regulates tumor angiogenesis. However, the results are controversial. This study investigated the effect and the mechanism of NO derived from eNOS on tumor angiogenesis. Methods: C57BL/6 mice injected with Lewis lung cancer cells were randomized into three groups. In the NO group, mice were injected with lung cancer cells transfected with the eNOS gene. Tumor-bearing mice were treated with N(G)-nitro-L-arginine methyl ester (L-NAME), an eNOS antagonist, in the L-NAME group, or with normal saline in the control group. The plasma concentration of NO and the number of endothelial progenitor cells (EPCs) In peripheral blood were detected. To elucidate the Involved mechanism, tumor vessel density, CD133+ cells and the expression of VEGF-VEGFR in tumors were also measured. Results: At 4 weeks after the injection of the Lewis cells, tumor volume in the control group was (3022 ± 401) mm3, while the tumor volumes were (1204 ± 97) mm3 and (1824 ± 239) mm3 in the L-NAME group and the eNOS group, respectively (P < 0.01). Proteins of eNOS and NO production increased significantly in Lewis lung cancer cells transfected with eNOS gene. But the number of CD133-positive cells and the vessel density in tumors were significantly lower in the eNOS group [(48 ± 19) per high-powered field (/HPF) and (19 ± 7)/ HPF, respectively] than in the control group (P < 0.05). There was no statistical difference in the number of EPCs in peripheral blood between each group. The concentration of NO In blood and tumor tissue was significantly decreased by L-NAME, while the tumor vessel density reduced to (12 ± 5)/HPF (P < 0.01 vs. the control group; P < 0.05 vs. the eNOS-transfected group). The number of EPCs In the blood and CD133-posltive cells In tumor tissue also decreased in the L-NAME group compared with the control group (P < 0.05). Conclusions: Our results demonstrate that NO derived from eNOS inhibits tumor growth via regulating angiogenesis, which may be due to its suppression on either the mobilization or homing of EPCs via VEGF binding to VEGFR.