Myocardial infarction (MI) is a cardiovascular disease with high morbidity and mortality. In this study, we focused on exploring the roles and underlying regulatory mechanisms of Hox transcript antisense intergenic RNA (HOTAIR) and miR-519d-3p in myocardial infarction. To comprehensively understand the role of microRNA in MI rat, we construct MI rat model by permanent ligation of the left anterior descending (LAD) coronary artery. Cardiac troponin I and creatine kinase-MB concentration measured by ELISA and infract size of heart section analyzed by TTC staining were served as evaluation indicators to confirmed the established model. Based on the bioinformatics assay and qRT-PCR, we found that the expression of miR-519d-3p was upregulated remarkably. Dual-luciferase reporter assays were performed to investigate the interaction of lncRNA HOTAIR and miR-519d-3p. In order to investigate the potential mechanism of lncRNA HOTAIR and miR-519d-3p, flow cytometry was applied to measure apoptotic cardiomyocytes and western blot was used to detect expressions of apoptotic related protein Bax, Bcl-2, and caspase-3 in cardiomyocytes in vitro and myocardial infraction in vivo. Downregulating miR-519d-3p or overexpressing HOTAIR alleviated MI or hypoxia-induced cardiomyocytes apoptosis. Taken together, our results showed that the interaction of miR-519d-3p and HOTAIR can protect MI and hypoxia-induced cardiomyocytes apoptosis, providing the potential therapeutic target for MI treatment.