Berberine is traditionally used to treat gastrointestinal (GI) motility disorders. The interstitial cells of Cajal (ICCs) are the pacemaker cells of the gastrointestinal tract, which are responsible for the production of gut movements. The present study aimed to investigate the effects of berberine on pacemaker potentials (PPs) in cultured ICC clusters from the mouse small intestine, and sought to identify the receptors involved and the underlying mechanisms of action. All experiments were performed on cultured ICCs, and a whole-cell patch-clamp configuration was used to record PPs from ICC clusters (current clamp mode). Under current clamp mode, berberine was shown to decrease the amplitude and frequency of PPs. However, these effects were suppressed by treatment with glibenclamide, a specific ATP-sensitive K+ channel blocker. Nor-binaltorphimine dihydrochloride (a kappa opioid receptor antagonist) did not suppress berberine-induced PP inhibition, whereas ICI 174,864 (a delta opioid receptor antagonist) and CTOP (a mu opioid receptor antagonist) did suppress the inhibitory effects of berberine. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor) or with KT-5720 (a protein kinase A inhibitor) did not suppress the effects of berberine; however, pretreatment with 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (a guanylate cyclase inhibitor) or KT-5823 [a protein kinase G (PKG) inhibitor] did. In addition, berberine stimulated cyclic guanosine monophosphate (cGMP) production in ICCs. These observations indicate that berberine may inhibit the pacemaker activity of ICC clusters via ATP-sensitive K+ channels and the cGMP-PKG-dependent pathway by stimulating mu and delta opioid receptors. Therefore, berberine may provide a basis for the development of novel agents for the treatment of GI motility dysfunction.