Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acupuncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and elecpuncture significantly reduced mechanical hypersensitivity following chronic constriction injury, especially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture significantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuropathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The results of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function.