Objective To investigate the role of membrane stretch on the pacemaker current in cultured interstitial cells of Cajal. Methods ICC was isolated from murine small intestine and then cultured in the medium with stem cell factors. After 72 hours, ICC was identified by immunohistochemical technique with monoclonal antibody for labeling c-kit protein and the pacemaker current in intestinal ICC were recoded under the conventional whole-cell patch clamp configuration. The membrane stretch was achieved by the application of direct pressure through the patch pipette and perfusion of hypotonic solution. Results At the 72nd hour, 2 to 4 projections were stacked out from the body of ICC, which interconnected with neighboring cells and formed cell networks under light microscope. Immunohistochemistry showed that c-kit protein was positively expressed on the surface of ICC. The spontaneous and rhythmic inward currents (pacemaker currents) were elicited under cells were voltage clamped in the whole-cell patch-clamp configuration and held at -60 mV. The sustained inward current was activated and the amplitudes and frequencies of pacemaker currents increased by the application of membrane stretch. Conclusion The mechanical stretch can regulate basic tone and frequency of gastrointestinal smooth muscle motility via activating or potentiating ICC pacemaker currents.