Objective To observe the effects of electroacupuncture (EA) on morphine-induced microglia activation and analgesic tolerance, and explore the potential mechanism of EA in the treatment of morphine analgesic tolerance. Methods A total of 60 clean-grade SD rats were randomly assigned to control group, morphine group, morphine＋EA group, and morphine＋EA＋colony-stimulating factor 1 (CSF1) group, with 15 rats in each group. Morphine analgesic tolerance model was established by continuous 7-d intrathecal injection of morphine in the morphine, morphine＋EA and morphine＋EA＋CSF1 groups. EA was given in the rats of morphine＋EA and morphine＋EA＋CSF1 groups at “Zusanli” and “Sanyinjiao” acupoints, with dilatational wave, frequency of 2/100 Hz, stimulation intensities of 0.5, 1.0, and 1.5 mA (10 min per intensity), once a day, for 7 consecutive days. Rats in morphine＋EA＋CSF1 group were given intrathecal injection with recombinant CSF1 protein for 7 consecutive days. The effect of EA on morphine analgesic tolerance in rats was observed by mechanical withdrawal threshold (MWT). After 7 d, the rats were sacrificed, and the L_4-6 spinal dorsal horn and dorsal root ganglion tissues were isolated. The expression of CSF1 protein and mRNA in the dorsal root ganglia and spinal dorsal horn was detected by Western blotting and quantitative polymerase chain reaction. The expression of ionized calcium-binding adapter molecule 1 (IBA-1), a marker of microglia in the spinal dorsal horn, was detected by immunofluorescence method, and the expression of interleukin (IL)-1β，IL-6 and tumor necrosis factor (TNF)-α in the spinal cord was detected by enzyme-linked immunosorbent assay (ELISA). Results After intrathecal injection of morphine, the percentage of maximal possible potential effect (%MPE) in the morphine group was decreased progressively, indicating that the morphine analgesic tolerance model was successfully constructed. Compared with the morphine group, the %MPE in the morphine＋EA group was increased after intrathecal injection at 3, 5 and 7 d (all P＜0.05). Compared with the morphine＋EA group, the %MPE in the morphine＋EA＋CSF1 group was all decreased after intrathecal injection at 3, 5 and 7 d (all P＜0.05). Compared with the control group, the expression of CSF1 protein and mRNA in dorsal root ganglion and the expression of CSF1 protein in spinal dorsal horn in the morphine group were increased (all P＜0.05). Compared with the morphine group, the expression levels of CSF1 protein and mRNA in dorsal root ganglion and CSF1 protein in spinal dorsal horn in the morphine＋EA group were decreased (all P＜0.05). There was no significant difference in the expression of CSF1 mRNA in the spinal dorsal horn among those groups (all P＞0.05). Compared with the control group, the expression of IBA-1 in the spinal dorsal horn of the morphine group was increased (P＜0.05). Compared with the morphine group, the expression of IBA-1 in the spinal dorsal horn of the morphine＋EA group was decreased (P＜0.05). Compared with the morphine＋EA group, the expression of IBA-1 in the spinal dorsal horn of the morphine＋EA＋CSF1 group was increased (P＜0.05). Conclusion EA can inhibit the activation of microglia in the spinal dorsal horn of rats and improve morphine analgesic tolerance in rats. The mechanism may be related to the reduction of CSF1 protein expression in the spinal dorsal horn.