Our study aims to excavate the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in myocardial infarction (MI), especially in an ischemia/reperfusion injury model and the underlying mechanism involving the MALAT1-miR144 axis. Our results demonstrated that the expression of MALAT1 has a higher level, while miR-144 expression significantly reduced in myocardial tissue after MI and also in left anterior descending (LAD)-ligation mice. This result was confirmed in vitro studies in HL-1 cardiomyocytes followed with hypoxia/reoxygenation. In addition, overexpression of MALAT1 by MALAT1-pcDNA injection into the mice with LAD increased myocardial apoptosis in vivo, while this effect was attenuated by miR-144 mimic. Bioinformatics analysis exhibits that 3′-UTR of MALAT1 is targeted to the miR-144-3p. Up-regulation miR-144 blunted the hypoxia- or MALAT1-induced cell apoptosis. In conclusion, the expression of MALAT1 was increased, whereas miR-144 expression was down-regulated in the myocardium after AMI. MALAT1 up-regulation plays a critical role in promoting cardiomyocytes apoptosis via targeting miR-144.