Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. In the present study, we investigated the effect of mitochondrial ATP-sensitive potassium (mitoKATP) channel on pacemaking activity in cultured ICCs from murine small intestine by using whole-cell patch clamp techniques. Under current clamp mode, at 10μM glibenclamide, there was no change in pacemaking activity of ICCs. At 30μM glibenclamide, an inhibitor of the ATP sensitive K⁢ channels, we could find two examples. If pacemaking activity of ICCs was irregulating, pacemaking activity of ICCs was changed into regulating and if in normal conditions, membrane potential amplitude was increased. At 50μM glibenclamide, the resting membrane potential was depolarized. At 3mM 5-HDA, an inhibitor of the mitoKATP channels, inhibited the pacemaking activity of ICCs. Both the amplitude and the frequency were decreased. At 5 mM 5-HDA, both the amplitude and the frequency were completely abolished. Diazoxide, an opener of the mitoKATP channels, was applied to examine its effect on pacemaking activity of ICCs. At 50μM concentration, the pacemaking activity of ICCs was inhibited. Both the amplitude and the frequency were decreased. At 1 mM concentration, both the amplitude and the frequency were completely abolished and the resting membrane potential was shaked. These results indicate that mitoKATP channel has an important role in pacemaking activity of ICCs.