To evaluate the analgesic effects of cinobufagin (CBG) on cancer-induced bone pain in rat and study the role of the muscarinic receptor M4 subtype (M4 mAChR) in its involvement. A total of 100 Female Sprague-Dawley rats were randomly divided into 5 groups (20): Sham group (group S), Cancer group (group A), Normal saline + CBG vehicle solution group (group ANS), Normal saline + 1 mg/kg CBG group (group ANC) and Tropicamide + 1 mg/kg CBG group (group ATC). Rats in group S were injected 10 μl Hank's solution into the left tibia medullar cavity, while rats in group A, ANS, ANC, and ATC were injected Walker 256 mammary cancer cells (10 μl, 2×10(7) cells/ml) into the same place. On day 9 post-inoculation rats in group ANS, ANC, and ATC were respectively received Saline (0.9%, 15 μl, i.t.), Saline (0.9%, 15 μl, i.t.)and 10 nmol of M4 mAChR blocker Tropicamide. After 10 min, ANS group, ANC group and ATC group were intraperitoneally injected with CBG vehicle solution, 1 mg/kg CBG and 1 mg/kg CBG. Model rats in each group were tested three times average as its basis pain threshold before injection cancer cells (T(0)). Mechanical withdrawal thresholds were measured on left hind paws, before 20 min (T(1)) and after 10 min (T(2)), 30 min (T(3)), 60 min (T(4)), 90 min (T(5)) and 120 min (T(6)) intrathecal injection. Left L4-L6 spinal dorsal horn and DRG were removed for determination of the expression of CaM-dependent kinaseⅡa (CaMKⅡa) and pCaMKⅡa by Western Blot after 60 min drug delivery. At each time point from T(1) to T(6), the mechanical pain thresholds of group S were (8.69±0.45), (8.63±0.44), (8.65±0.39), (8.84±0.23), (8.80±0.14), (8.75±0.14) g, respectively, and the mechanical pain thresholds of group A were (6.37±0.30), (6.42±0.13), (6.29±0.17), (6.25±0.22), (6.34±0.33), (6.36±0.34) g, the difference was statistically significant (-16.41, -23.47, -30.25, -17.35, -19.52, -22.56, all 0.01). At each time point from T(3) to T(5), the mechanical pain thresholds of the ANS group were (6.42±0.32), (6.39±0.34), (6.26±0.32) g, respectively, and the mechanical pain thresholds of the ANC group were (7.29±0.34), (7.81±0.15), (7.54±0.19) g, the difference was statistically significant (13.52, 14.22, 17.33, all 0.01). At each time point from T(3) to T(5), compared with the ANC group, the mechanical pain threshold of the ATC group decreased (6.55±0.23), (6.84±0.46), (6.80±0.43) g, and the difference was statistically significant (-12.69, -11.26, -10.33, all 0.01). At the time of T(4), the expressions of pCaMKⅡa in the spinal dorsal horn of each group were (0.67±0.05), (1.64±0.12), (1.57±0.14), (0.78±0.09), (1.39±0.11), respectively, and the expressions of pCaMKⅡa in DRG of each group were (1.65±0.39), (3.59±0.17), (3.43±0.32), (2.17±0.34), (2.95±0.23). The differences were statistically significant (179.89, 198.76, both 0.01). Compared with the S group, the expression of pCaMⅡa was up-regulated in group A. Compared with ANS group, the expression of pCaMKⅡ a was down-regulated in ANC group. Compared with ANC group, the expression of pCaMK Ⅱ a was up-regulated in ATC group. The expression of CaMKⅡa in spinal dorsal horn and DRG was not statistically significant (1.25, 2.79, both 0.05). These results demonstrated that M4mAChR participated in mediating the alleviation of hyperalgesia by cinobufagin in rats with bone cancer pain, and its mechanism may be related to pCaMKⅡa/CaMKⅡa signaling pathway.