Objective:To observe the effect of botulinum toxin A (BTX-A) on pain, the activation of microglia and the expression of tumor necrosis factor-α (TNF-α) in the spinal cord in arthritis, and to explore how BTX-A treatment relieves pain.Methods:Sixty clean, male Sprague-Dawley rats were randomly divided into a sham operated group, a Freund′s adjuvant group and a BTX-A group. The ankle cavities of the left hind limbs of all of the rats except those in the sham group were injected with 50μl of Freund′s adjuvant to establish an arthritis pain model. The sham operated group received 50μl of saline solution as controls. Afterward the sham operation group and the Freund′s adjuvant group were given another 20μl of normal saline, while the BTX-A group was injected with 20μl of botulinum toxin A, again into the ankle joint cavity of the left hind limb. The mechanical and thermal pain thresholds of the rats in each group were measured 1 day before the modeling and 1, 3, 7, 14 and 21 days afterward. Western blotting and immunofluorescence staining were used to detect the expression of IBA-1 and IBA-1-IR. In addition, the expression of TNF-α protein and TNF-α mRNA in the spinal cord was detected using ELISA and RT-PCR.Results:Compared with the Freund′s adjuvant group, the latency of the mechanical and thermal pain thresholds had increased significantly in the BTX-A group after 3 days. The differences remained significant until the 14th day after the injection. The expression of IBA-1 protein and the number of immunopositive cells in the spinal cord decreased significantly, as did the expression of TNF-α protein and mRNA.Conclusions:Botulinum toxin A can alleviate the pain induced by Freund′s adjuvant. The analgesic mechanism may be related to inhibiting the activation of spinal microglia and the release of TNF-α.