Cardiovascular ischemic disease refers to a large class of conditions that are harmful to human health. A number of previous studies have demonstrated that microRNAs (miRs) have notable roles in regulating cardiac injury. miR-144 is influential in the differentiation, growth, and metastatic processes of cells; however, the impact of miR-144 in cardiac ischemia/reperfusion (I/R) injury has not been thoroughly elucidated to date. In the present study, reverse transcription quantitative polymerase chain reaction was used to evaluate RNA expression. In addition, TTC staining was performed to detect the infarct area of the ischemic myocardia and a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay was utilized to detect the apoptosis of the myocardia. It was observed that miR-144 expression is downregulated in an I/R model in rats and that overexpression of miR-144 significantly reduced myocardial ischemic injury and apoptosis. Consistent with this result, similar findings were demonstrated in H9c2 cells subjected to hypoxia/reoxygenation. Bioinformatic analysis using MiRanda and TargetScan, and luciferase assays confirmed that forkhead box protein O1was the target of miR-144. These findings suggest that miR-144 may be exploited as a novel molecular marker or therapeutic target for myocardial I/R injury.