Abstract: Cionbufagin has anti-inflammatory and analgesic effects. It is of great value in the treatment of bone cancer pain, but its mechanism is still unclear. To generate a bone metastasis model of breast cancer, 4×10⁵ Walker-256 cells were inoculated into the left hind limb of SD rats. The experimental protocol was approved by the Medical Laboratory Animal Ethics Committee of Medical College of China Three Gorges University. Rats were randomly divided into sham, model, cionbufagin, morphine, saline, minocycline, microglia inhibitor (RS102895) and co-treatment with cionbufagin and minocycline group. The cionbufagin (5 mL·kg^-1, i.p.), morphine (8 mg·kg^-1, i.p.) and co-treatment groups (included cionbufagin 5 mL·kg^-1, i.p.) received continuous administration from day 9 to day 21. The saline, minocycline (2.5 μg·μL^-1, 20 μL), RS102895 (1.5 μg·μL^-1, 20 μL) and co-treatment groups (included minocycline 2.5 μg·μL^-1, 20 μL) received continuous administration by intrathecal cannulation from day 12 to day 21. Bone destruction of the left hind limb of rats was detected by hematoxylin-eosin staining (H&E). The pain threshold before treatment and at day 2, 5, 7, 9, 12, 14, 17 and 20 was measured by behavioral indexes. Activation and expression of a microglia marker (Iba-1) was determined by immunofluorescence and Western blot. The level of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) in rat spinal cord was measured by enzyme-linked immunosorbent assay (ELISA). H&E results showed that cionbufagin effectively inhibited the destruction of bone marrow in rats with bone cancer pain; cionbufagin treatment significantly increased the mechanical and thermal pain threshold. Immunofluorescence showed that cionbufagin effectively inhibited the activation of microglia in the spinal dorsal horn. Western blot analysis confirmed that the activation of microglia in the spinal dorsal horn was inhibited by cionbufagin treatment. It was also found that the CCL2/CCR2 pathway may be involved in the analgesic effect of cionbufagin. These results suggest that cionbufagin can effectively alleviate bone cancer pain, possibly by inhibiting the release of inflammatory factors and the activation of spinal microglia cells through the CCL2/CCR2 pathway.