We evaluated miRNA-499′s effect on myocardial ischemia reperfusion (IR) injury. Serum Creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) levels, myocardial infarction area, miRNA-499, Bak1, PI3K, Akt Bcl-2, and Bax protein expressions were analyzed. CK levels in sham operation group were lowest after ischemia, while IR and negative control groups were highest, followed by the miR-499 group. IR and negative control groups showed highest MDA level, and lowest SOD, GSH and GSH-Px, followed by the miR-499 group. The IR group and negative control group showed significantly more infarct area than that of miR-499 group (p< 0.05). The lowest levels of Bak1, PI3K and Akt proteins were found in sham operation and miR-499 group, and the opposite was found in IR group (p< 0.05). Bcl-2 and Bax level expressions in sham-operation group and miR-499 group were lowest (p<0.05). Bak1 is a target for miR-499, and fluorescence intensity of mutant plasmid was increased (p< 0.05). In conclusion, miRNA-499 specifically binds to the target gene Bak1 and down-regulates Bak1 gene to activate the PI3K/Akt signaling pathway, therefore inhibiting myocardial apoptosis and ultimately ameliorating myocardial IR injury.