The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ + LPS) for 24 h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca2+ oscillation in the presence of nifedipine. Treatment with IFN-γ + LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ + LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ + LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ + LPS. Treatment with IFN-γ + LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ + LPS-treated clusters, suggesting IFN-γ + LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ + LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.