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. In summary, inhibition of miR‐327 may alleviate I/R‐induced myocardial injury and suppress both extrinsic and intrinsic apoptotic cascades after myocardial ischemia/reperfusion injury (MI/RI) by targeting ARC. The manipulation of miR‐327‐mediated ARC stability may offer a new therapeutic strategy for MI/RI.