Apoptosis is the major cause of cardiomyocyte death in myocardial ischemia/reperfusion injury (MI/RI). Increasing evidence suggests that microRNAs (miRNAs) can contribute to the regulation of cardiomyocytes apoptosis by posttranscriptional modulation of gene expression networks. However, the effects of miR-327 in regulating MI/RI-induced cardiomyocytes apoptosis have not been extensively investigated. This study was performed to test whether miR-327 participate in cardiomyocytes apoptosis both in vitro and in vivo, and reveal the potential molecular mechanism of miR-327 regulated MI/RI through targeting apoptosis repressor with caspase recruitment domain (ARC). Sprague-Dawley (SD) rats were subjected to MI/RI by left anterior descending coronary artery occlusion for 30 min and reperfusion for 3 hr. H9c2 cells were exposed to hypoxia for 4 hr and reoxygenation for 12 hr to mimic I/R injury. miRNA-327 recombinant adenovirus vectors were transfected into H9c2 cells for 48 hr and rats for 72 hr before H/R and MI/RI treatment, respectively. The apoptosis rate, downstream molecules of apoptotic pathway, and the target reaction between miRNA-327 and ARC were evaluated. Our results showed that miR-327 was upregulated and ARC was downregulated in the myocardial tissues of MI/RI rats and in H9c2 cells with H/R treatment. Inhibition of miR-327 decreased the expression levels of proapoptotic proteins Fas, FasL, caspase-8, Bax, cleaved caspase-9, cleaved caspase-3, and the release of cytochrome-C, as well as increasing the expression levels of antiapoptotic protein Bcl-2 via negative regulation of ARC both in vivo or vitro. In contrast, overexpression miR-327 showed the reverse effect. Moreover, the results of luciferase reporter assay indicated miR-327 targets ARC directly at the posttranscriptional level. Taken together, inhibition of miR-327 could attenuate cardiomyocyte apoptosis and alleviate I/R-induced myocardial injury via targeting ARC, which offers a new therapeutic strategy for MI/RI.