Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of caffeic acid phenethyl ester (CAPE) on the pacemaker potentials of ICCs from the mouse small or large intestine. Using the whole-cell patch-clamp configuration, we found that CAPE depolarized the pacemaker potentials of cultured ICCs from the murine small intestine in a dose-dependent manner. The estrogen receptor (ER) β antagonist PHTPP completely inhibited CAPE-induced depolarization, but the ERα antagonist BHPI did not. Intracellular GDP-β-S and pretreatment with Ca2+-free solution or thapsigargin also blocked CAPE-induced depolarization. To investigate the mechanisms of CAPE-mediated depolarization of ICCs, we used the nonselective cation channel (NSCC) inhibitor flufenamic acid, the Cl– channel blocker, mitogen-activated protein kinase (MAPK) inhibitors PD98059, SB203580, or SP600125, and PI3 kinase inhibitor LY294002. All inhibitors blocked the CAPE-induced pacemaker potential depolarization of ICCs. These results suggest that CAPE induces pacemaker potential depolarization through ERβ in a G protein, NSCC, Cl– channel, MAPK- and PI3 kinase dependent manner via intracellular and extracellular Ca2+ regulation in the murine small intestine. CAPE may therefore modulate GI motility by acting on ICCs in the murine small intestine.