<p>Ring finger protein 146 (RNF146) participates in regulating ferroptosis and ferroptosis is involved in myocardial ischemia/reperfusion injury (MI/RI). However, the effects and mechanisms of RNF146 in MI/RI are still unclear. TTC, H&amp;E, IHC, DHE stainings, and echocardiography technology were used to determine the myocardial infarction area, pathological injury, level of RNF146, ROS, and cardiac function parameters, respectively. CCK-8 was employed to determine the cell viability. The corresponding kits, RT-qPCR, and western blot were adopted to determine the levels of CK-MB, LDH, Fe²⁺, MDA, ROS, gene expression levels of RNF146 and death-associated protein kinase 1 (DAPK1), protein expression levels of RNF146, DAPK1, GPX4, FTH1, and ACSL4. Co-immunoprecipitation, cycloheximide tracking, and ubiquitination assays to investigate the relationship between RNF146 and DAPK1. Ferroptosis occurred in mice with MI/RI and inhibiting ferroptosis could alleviate MI/RI. Moreover, the expression of RNF146 is down-regulated in MI/RI, and overexpression of RNF146 can inhibit H/R-induced ferroptosis of cardiomyocytes. Mechanistically, RNF146 promotes ubiquitination and degradation of DAPK1. In addition, the effects of overexpressed RNF146 in alleviating MI/RI were effectively reversed by overexpressing DAPK1. This study demonstrated that RNF146 alleviates MI/RI by facilitating the ubiquitylation-mediated degradation of DAPK1 to reduce ferroptosis.</p>