Objective To investigate the underlying mechanisms of the analgesic effect of electroacupuncture (EA), we assessed the effect of EA on microglial activation and the expression of brain-derived neurotrophic factor (BDNF) in the L₄-L₆ spinal cord in rats with diabetic neuropathic pain (DNP). Methods SD rats were divided into a normal group (N group), a model group (M group), and an EA group (EA group). The rats in the EA group were treated with EA at “Zusanli” (ST36) and “Kunlun” (BL60) starting on week 6, with 30 min per session, once every other day for 7 sessions. Fasting blood glucose ( FBG), thermal paw withdrawal latency ( PWL), and paw withdrawal thresholds (PWT) were measured at baseline, 4 weeks, 6 weeks, and 8 weeks. Immunofluorescence was used to measure the expression of microglia (CD11b) and BDNF in samples from the L₄-L₆ spinal cord. Results 1) Compared with that in the N group, FBG was significantly increased in the M group and the EA group, and there was no significant difference in FBG between the M group and the EA group at 6 weeks and 8 weeks.(2) Compared with that in the N group, the PWL and PWT were significantly decreased in the M group at 6 weeks and 8 weeks. After EA treatment, compared with that in the M group, the PWL and PWT in the EA group were significantly increased at 8 weeks. there was no significant difference in PWL and PWT between the M group and the EA group at 6 weeks. (3) Compared with that in the N group, the expression of microglia (CD11b) and BDNF in the M group were increased significantly, and compared with that in the M group, the expression of microglia ( CD11b) and BDNF were significantly decreased in the EA group. Conclusions The result suggest that microglia and BDNF in the spinal cord might be involved in the development of DNP. The analgesic effect of EA might be achieved by inhibition of the expression of microglia and BDNF.