The Citrus unshiu peel has been widely used for the treatment of gastrointestinal (GI) disorders in Eastern traditional medicine. The present study aimed to investigate the effects of Citrus unshiu peel extract (CPE) on the pacemaker activity of the GI tract in cultured interstitial cells of Cajal (ICCs) derived from the mouse small intestine. The whole‑cell patch‑clamp configuration was used to record pacemaker potentials. In current clamp mode, exposure to CPE caused membrane pacemaker depolarization in a concentration‑dependent manner. In the presence of the muscarinic M2 receptor antagonist, methoctramine, CPE induced membrane pacemaker depolarization, whereas treatment with the muscarinic M3 receptor antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide, inhibited CPE‑induced responses. When the pipette solution contained guanosine 5'-(β-thio) diphosphate trilithium salt (1 mM), CPE marginally induced membrane pacemaker depolarization. In addition, CPE‑induced membrane pacemaker depolarization was inhibited following exposure to the active phospholipase C (PLC) inhibitor U‑73122, but not the inactive PLC inhibitor U‑73343. In the presence of a p42/p44 mitogen‑activated protein kinase (MAPK) inhibitor (PD98059), a p38 MAPK inhibitor (SB203580) or a c‑jun NH2‑terminal kinase (JNK) II inhibitor, CPE failed to induce membrane pacemaker depolarization. These results suggest that CPE may affect GI motility through modulating ICC pacemaker activity by activating the muscarinic M3 receptor and inducing the G‑protein dependent PLC and MAPK signaling pathways.