MicroRNA-34a (miR-34a) promotes apoptosis via down-regulating many anti-apoptotic proteins. Aldehyde dehydrogenase 2 (ALDH2) is an anti-apoptotic enzyme whose activity decline associates with myocardial injury. We tested hypothesis that miR-34a might play a pro-apoptotic role in myocardial infarction (MI) by down-regulating ALDH2. MiR-34a was highly increased while ALDH2 expression was decreased after experimental MI. Overexpression of miR-34a in neonatal rat cardiomyocyte could significantly enhance apoptosis and down-regulate ALDH2 expression. In 293 cells, luciferase reporter assay results demonstrated that ALDH2 was a direct target of miR-34a. Serum miR-34a levels in acute myocardial infarction (AMI) patients and rats were significantly higher than healthy subjects and sham rats. Our results proved that miR-34a could promote cardiomyocyte apoptosis via negatively regulating ALDH2 and circulating miR-34a was increased in the condition of MI. Thus, miR-34a may constitute a new therapeutic target and diagnostic marker for patients with MI.