Ischemic heart disease often causes heart failure. During ischemia-reperfusion (I/R), excessive reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are produced in cardiac tissue, where they induce cell death. We previously reported that a TRPM4 channel inhibitor 9-Phenanthrol protects cardiac I/R injury in the excised rat heart. Based on this finding, we hypothesized that TRPM4 channels are involved in the pathophysiology of the cardiac I/R injury. We confirmed that intravenous application of 9-Phenanthrol mitigated the development of myocardial infarction caused by the occlusion of the left anterior descending artery in rats. Positive expression of TRPM4 channels in the ventricular cardiomyocytes was confirmed by immuno-histochemistry. To evaluate the toxic effect of ROS on cardiac cells, we measured cellular viability of H9c2 cardiomyocytes underwent H2O2 challenge. Pretreatment of 9-Phenanthrol preserved cellular viability. Furthermore, knockdown of TRPM4 channels preserved the viability of H9c2 cardiomyocytes exposed to H2O2. These results suggest that TRPM4 channels are involved in development of cardiac ischemia-reperfusion injury.