Emerging literature suggests that pyroptosis plays a critical role in ischemia/hypoxia (I/R) -induced myocardial injury. Melatonin has been implicated in attenuating I/R-induced injury of cardiomyocytes. Nevertheless, whether melatonin inhibits I/R-induced pyroptosis of cardiomyocytes and the underlying molecular mechanisms remain unexploited.H9c2 cardiomyocytes were cultured under oxygen-glucose deprivation/reperfusion (OGD/R) condition to establish a myocardial pyroptosis model in vitro. OGD/R-induced pyroptosis was evaluated by CCK-8 assay, IL-1β and IL-18 release, and western blotting. Luciferase reporter assay was utilized to validate the association between miR-155 and Forkhead box O3a (FOXO3a).Melatonin could inhibit OGD/R-induced pyroptosis of H9c2 cells and upregulation of FOXO3a contributed to the antipyroptotic effect of melatonin. Melatonin reduced miR-155 expression, which led to FOXO3a upregulation and inhibition of pyroptosis in OGD/R-exposed H9c2 cells. miR-155 inhibitor enhanced the antipyroptotic effect of melatonin in OGD/R-exposed H9c2 cells. Melatonin-induced downregulation of miR-155 and upregulation of FOXO3a were reversed by melatonin receptor 2 (MT2) siRNA. Melatonin treatment also led to an increased level of apoptosis repressor with caspase recruitment domain (ARC), which was inhibited by FOXO3a siRNA. Moreover, silencing ARC by siRNA significantly blocked the antipyroptotic actions of melatonin, whereas ARC overexpression enhanced the antipyroptotic actions of melatonin in OGD/R-exposed H9c2 cells.Our findings demonstrated that melatonin prevented OGD/R-induced pyroptosis via regulating the MT2/miR-155/FOXO3a/ARC axis in cardiomyocytes.