Objective To explore the effect and mechanism of electroacupuncture on eNOS in mobilizing endothelial progenitor cells (EPCs) in rat bone marrow and peripheral blood to promote revascularization in focal cerebral ischemia/reperfusion rat. Methods A total of 100 healthy male adult Sprague-Dawley (SD) rats were randomly divided into normal group (N), model group (I/R), electroacupuncture group (I/RE) and I/RE plus L-NAME (A specific antagonist of eNOS) group (I/REL), and were further divided into 1 d, 2 d and 7 d subgroups after reperfusion, 10 rats in each group, in addition to the N group. The rats received filament occlusion of the right middle cerebral artery for 1.5 hours followed by reperfusion. “Baihui” (GV 20)/“Siguan” (Hegu LI 4/Taichong LR 3) were selected as acupucture points. Flow cytometer was used to detect the percentage of EPCs in bone marrow and peripheral blood. The expression of VEGFR2 mRNA was tested by fluorescence quantitative PCR. Immunohistochemical staining was used to detect VEGFR2⁺ cells and to stain the CD34⁺microvessels. Results Compared with the I/R group, there was a significant up-regulation of the percentage of EPCs in bone marrow and peripheral blood by electroacupuncture(P<0.01,P<0.05), but decreased by the inhibitor of eNOS (P<0.01). Compared with the I/R group, the VEGFR2⁺cells, expression of VEGFR2 mRNA and CD34⁺microvessels were significantly increased in the I/RE group (P<0.01), but decreased in the I/REL group (P<0.01).Conclusions Electroacupuncture can effectively mobilize EPCs to promote the revascularization in focal cerebral ischemia/reperfusion rat. This effect is attenuated by inhibitor of eNOS, suggesting that the activation of eNOS mediated by electroacupuncture may be related to mobilizing EPCs to promote the revascularization.