The aim of this study was to explore the role and regulatory mechanism of microRNA-298 (miR-298) in myocardial ischemic injury. H9c2 cardiomyocytes were cultured under hypoxia (3 % O₂) conditions to induce myocardial ischemic injury. Subsequently, the effects of miR-298 overexpression and suppression on hypoxia-induced myocardial damage in H9c2 cells were investigated. Moreover, the target of miR-298 was identified in H9c2 cells and the relationship between miR-298 and the activation of PTEN/PI3K/AKT signaling pathway was explored. miR-298 was upregulated in hypoxia-stimulated H9c2 cells. Overexpression of miR-298 distinctly aggravated hypoxia-induced myocardial damage in hypoxia-treated H9c2 cells, whereas inhibition of miR-298 alleviated hypoxia-induced injury. Moreover, miR-298 negatively regulated the expression of cyclin D1, and cyclin D1 was a target of miR-298 in H9c2 cells. Suppression of cyclin D1 significantly reversed the effects of suppression of miR-298 on hypoxia-induced myocardial damage. Lastly, inhibition of miR-298 activated the PTEN/PI3K/AKT signaling pathway, and this effect could be reversed after suppression of cyclin D1. Our results reveal that miR-298 may exacerbate myocardial ischemic injury by targeting cyclin D1 and regulating the activation of PTEN/PI3K/AKT signaling pathway. miR-298 may serve as a promising targets for reducing myocardial ischemic injury.