Cancer-induced bone pain (CIBP) is a common pain in clinics, which can reduce the quality of life and increase the mortality of patients, but the treatment of CIBP is limited. This study was designed to investigate the analgesic effect of α-cobratoxin on CIBP and further to explore the molecular target and potential signal pathway. As shown by the mechanical allodynia test in a CIBP rat model, administration of α-cobratoxin produced significant analgesia in a dose-dependent manner, and the analgesic effects were blocked by pretreatment with an intrathecal injection of M4 mAChR-siRNA or intraperitoneal injection of tropicamide, an antagonist of M4 muscarinic cholinergic receptor. Whole-cell patch-clamp recording showed that α-cobratoxin can decrease the spontaneous firing and spontaneous excitatory postsynaptic currents of SDH neurons in CIBP rats. In primary lumber SDH neurons, intracellular calcium measurement revealed that α-cobratoxin decreased intracellular calcium concentration, and immunofluorescence demonstrated that M4 muscarinic cholinergic receptor and CaMKII/CREB were co-expressed. In the CIBP model and primary SDH neurons, Western blot showed that the levels of p-CaMKII and p-CREB were increased by α-cobratoxin and the effect of α-cobratoxin was antagonized by M4 mAChR-siRNA. The quantitative polymerase chain reaction (qPCR) results showed that α-cobratoxin downregulated the expression of proinflammatory cytokines through M4 muscarinic cholinergic receptor in SDH. These results suggest that α-cobratoxin may activate M4 muscarinic cholinergic receptor, triggering the inhibition of SDH neuronal excitability via CaMKII signaling pathway, thereby resulting in antagonistic effects in the CIBP rat model.