Abstract: Objective To evaluate the myocardial protective effect of semaglutide on ischemia-reperfusion (I/R) injury in mice and investigate its potential mechanism.Methods C57BL/6J mice were used to establish an I/R injury model, and divided into three groups: Sham (control), I/R (ligation of the left anterior descending coronary artery for 45 min followed by 24 h of reperfusion), and I/R+Semaglutide (I/R+Sem). HL-1 mouse cardiomyocytes were cultured in vitro to establish a hypoxia/reoxygenation (H/R) injury model, and divided into three groups: Control, H/R (4 h of hypoxia followed by 4 h of reoxygenation), and H/R+Semaglutide (H/R+Sem, 5 mmol/L). Mouse cardiac function and myocardial infarction area were evaluated using echocardiography and TTC staining. Myocardial fibrosis was assessed by Hematoxylin and Eosin (HE) staining, Masson's trichrome staining, and immunofluorescence staining. Mitochondrial morphology was observed by transmission electron microscopy. The levels of Fe²⁺, glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured in mouse myocardial tissue and HL-1 cells. The expression of glutathione peroxidase-4 (GPX4) and cyclooxygenase-2 (COX2) was detected by Western blot. Differential gene expression was analyzed by RNA sequencing (RNA-seq) and quantitative real-time PCR (RT-qPCR). The effect of S100 calcium binding protein A9 (S100A9) overexpression on the protein kinase C(PKC)-S100A9 pathway in cardiomyocytes after H/R injury was assessed.Results Semaglutide reduced myocardial injury caused by I/R, promoted the recovery of cardiac function, alleviated myocardial fibrosis, inhibited ferroptosis in cardiomyocytes, and improved oxidative stress response. RNA-seq results indicated that S100A9 is a potential target gene of semaglutide in protecting against I/R injury. Semaglutide exerted its cardioprotective effect by activating the PKC signaling pathway, which decreased the expression of S100A9.Conclusions Semaglutide protects against myocardial ischemia-reperfusion injury by inhibiting ferroptosis in cardiomyocytes. The underlying mechanism may involve the activation of the PKC signaling pathway and the reduction of S100A9 expression.