Abstract: Objective To investigate the impact of microRNA-323（miR-323） on cardiomyocyte injury induced by ischemia-reperfusion. Methods The H9C2 cell models for hypoxia/reoxygenation（HO） injury and mice models for ischemia/reperfusion（I/R） were established. Cell viability was assessed through methyl thiazolyl tetrazolium（MTT） colorimetry, while cardiomyocyte injury was determined using lactate dehydrogenase（LDH） release assays. Cardiac function was evaluated via ultrasound and the myocardial infarction area was measured through 2,3,5-triphenyl-2H-tetrazolium chloride-evans blue（TTC-EB） double staining. The protein levels of prostate apoptosis response-4（PAR4） and cleaved caspase-3 were assessed via Western-blot in H9C2 cell models both pre-and post-hypoxia/reoxygenation. Results In the cellular model, after hypoxia/reoxygenation, the HO-miR-323 group demonstrated a substantially reduced LDH content（t=13.65, P＜0.01） compared to the control group, concurrently leading to a noteworthy amelioration in cell viability（t=8.50, P＜0.01）. In contrast, the HO-miR-323-inhibitor group manifested a notable increase in LDH content（t=22.75, P＜0.01） and a significant reduction in cell viability（t=8.46, P＜0.01）. In the animal experiment, miR-323-agomiR significantly improved cardiac function compared to the negative control（NC） group left ventricular ejection fraction（LVEF）:（39.9±4.4）% vs（32.0±3.2）%, t=3.54, P＜0.01; left ventricular fractional shortening（LVFS）:（19.2±2.0）% vs（15.4±2.1）%, t=3.17, P=0.01. But miR-323-antagomiR reversed this improvement LVEF:（29.2±3.7）% vs（34.0±4.1）%, t=2.20, P=0.04; LVFS:（11.9±1.7）% vs（14.9±1.9）%, t=2.74, P=0.01. Furthermore, compared to the control group, the miR-323-agomiR group exhibited a significant reduction in the infarct size percentage （47.2±9.1）% vs（57.7±5.9）%, t=2.38, P=0.04 and relative risk area （30.2±4.2）% vs（44.9±6.4）%, t=4.70, P＜0.01. Conversely, the miR-323-antagomiR group demonstrated an opposite pattern, with an increase in infarction area （65.1±7.8）% vs（53.2±6.7）%, t=2.83, P=0.02 and the relative area at risk （53.5±4.5）% vs（44.3±7.5）%, t=2.59, P=0.03. Transfection of miR-323 inhibited the protein expression of PAR4 and cleaved caspase-3 simultaneously in H9C2 cell hypoxia/reoxygenation models. Conclusion miR-323 can ameliorate cardiomyocytes injury caused by ischemia/reperfusion and improve cardiac dysfunction by targeting PAR4 to regulate cleaved caspase-3.