Myocardial ischemia-reperfusion (I/R) injury is leading cause of death worldwide. miR-34a-5p was up-regulated in myocardial ischemia-reperfusion injury rats. We aim to explore how miR-34a-5p inhibition protected myocardium against I/R injury in both cell and animal models. In vivo rat and in vitro cell model were firstly constructed. Quantitative real-time polymerase chain reaction was employed to investigate expression of miR-34a-5p and its target genes. Functional assays were conducted to detect the impact of miR-34a-5p on myocardial I/R injury. Enzyme-linked immunosorbent assay was performed to validate the expression levels of marker proteins of ischemia-reperfusion I/R-induced myocardial injury. MTT was performed to assess the cell viability and flow cytometry was utilized to detect cell apoptosis and reactive oxygen species accumulation. The interaction between miR-34a-5p and Notch Receptor 1 were also examined through luciferase reporter assay. miR-34a-5p was up-regulated post-reperfusion at rat myocardium. miR-34a-5p inhibitor attenuated myocardial ischemia-reperfusion injury, as shown by decreasing apoptosis rate, reducing infarct size and reactive oxygen species accumulation. In in vitro cell model, miR-34a-5p inhibitor also promoted cell proliferation, inhibited cell apoptosis and reactive oxygen species accumulation through targeting Notch Receptor 1 signaling. Our results revealed that miR-34a-5p knocking down attenuated myocardial I/R injury by promoting Notch Receptor 1 signaling-mediated inhibition of reactive oxygen species accumulation and cell apoptosis. Hence, miR-34a-5p might be a potential target for treatment of myocardial ischemia-reperfusion injury.