Over the past few decades, most cardiac reperfusion research has focused on the pathogenesis of myocardial ischemia reperfusion injury (MIRI), while little attention has been paid to microvascular ischemia reperfusion (I/R) injury, and few strategies have been available to reverse this pathological process. c-Myc, a proto-oncogene, is related to cardiomyocyte apoptosis and inflammation. It has been reported that Sam68 is involved in inflammatory disorders. However, whether c-Myc and Sam68 can modulate pyroptosis in cardiac microvascular endothelial cells (CMECs) remains unknown.In this study, I/R models were established by ligation of the left anterior descending coronary artery (LAD) in rats and hypoxia-reoxygenation (H/R) in CMECs. The myocardial tissue structure, myocardial infarct size, microcirculation perfusion, cardiac function, and protein and RNA expressions were detected by hematoxylin and eosin (HE) staining, Evans blue and triphenyltetrazolium chloride (TTC) staining, gelatin-ink perfusion, echocardiography, Western blot, and real-time PCR, respectively.Our results demonstrated that c-Myc and Sam68 expressions were increased in I/R rats and H/R CMECs. In vitro studies confirmed that c-Myc inhibition suppressed H/R-induced pyroptosis in CMECs, and this effect was mediated through the Sam68/NF-κB pathway. In vivo, the small molecule c-Myc inhibitor 10058-F4 attenuated cardiac I/R injury, improved microvascular perfusion, and was associated with reduced endothelial pyroptosis.Our study uncovered a novel c-Myc/Sam68/NF-κB/pyroptosis regulatory axis in CMECs under I/R stress, and suggests that targeting this axis, and thereby protecting endothelial function, could be a promising strategy to improve the efficiency of reperfusion therapy in clinical situations.