α-cobratoxin (α-CbTX), a long-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia.However, to date, the relevant mechanisms are not completely understood.In the present study, we identified a novel functional role of α-CbTX in modulating T-type Ca2+ channel currents (T-currents) in small sensory neurons as well as pain behaviors in mice.We found that α-CbTX inhibited T-currents in a dose-dependent manner in mouse small dorsal root ganglia (DRG) neurons.α-CbTX at 0.5 μM reversibly inhibited T-currents by ~22.3%.This inhibitory effect was blocked by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M4 mAChR antagonist tropicamide, while naloxone, an opioid receptor antagonist had no effect.Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of α-CbTX.By using depolarizing prepulse, we observed the absence of functional coupling between G-protein βγ subunits and T-type Ca2+ channels.Pretreatment of the cells with H89, a protein kinase A (PKA) inhibitor, or intracellular application of PKI 6-22, abolished t-CbTX-induced T-current inhibition, whereas inhibition of PKC or phospholipase C (PLC) elicited no such effects.Intrathecal administration of α-CbTX (5 μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models.Moreover, α-CbTX-induced antinociceptions in mice could be abrogated by tropicamide.Taken together, these results suggest that u-CbTX inhibits small DRG neuron T-currents via a M4 mAChR-dependent PTX-sensitive PKA pathway, which could contribute to its analgesic effects in mice.