Background: Atractylodes japonica has been commonly used to treat gastrointestinal (GI) disorders in Korean traditional medicine. Interstitial cells of Cajal (ICCs) are pacemaker cells in the GI tract and can regulate GI motility. Objective: To investigate the effects of the extract of Atractylodes japonica (AJE) on pacemaker potentials generated by ICCs from murine small intestine. Materials and Methods: Enzymatic digestion was performed to dissociate ICCs. All experiments on ICCs were performed after 12 h of culture. The whole-cell patch-clamp configuration was used to record pacemaker potentials generated by ICC. Results: AJE (0.1—1 mg/mL) depolarized pacemaker potentials in a concentration-dependent manner and decreased the amplitudes of pacemaker potentials at all concentrations in the current-clamp mode. Pretreatment with Y25130 (a 5-HT3receptor antagonist), RS39604 (a 5-HT4receptor antagonist), or SB269970 (a 5-HT7receptor antagonist) had no effects on depolarization of pacemaker potentials induced by AJE. In addition, pretreatment with 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (a muscarinic M3receptor antagonist) inhibited AJE-induced depolarization of pacemaker potential; however, pretreatment with methoctramine (a muscarinic M2receptor antagonist) did not affect depolarization of pacemaker potentials induced by AJE. In the presence of an external Na+-free solution, the pacemaker potentials decreased, and under this condition, AJE did not depolarize the pacemaker potentials. Flufenamic acid, a nonselective cation channel (NSCC) blocker, decreased the pacemaker potential, which in turn inhibited AJE-induced depolarization of pacemaker potential. Conclusion: The results of this study suggest that AJE depolarized the pacemaker potentials generated by ICC by stimulating muscarinic M3receptors, but not 5-HT receptors, through NSCCs. Therefore, AJE can be a novel prokinetic agent.