Ischemic reperfusion (I/R) injury is a serious problem in the treatment of ischemic heart disease. MicroRNA-208a (miR-208a) is a cardiac-specific or cardiac-enriched miRNA. This study was aimed to assess the role of miR-208a in I/R injury. H9c2 cells were used to simulate I/R injury in vitro. miR-208a expression level was measured by qPCR. H9c2 cells after simulated I/R injury were transfected with miR-208a mimic, AOS-miR-208a or negative controls. LDH release, MDA and SOD contents were measured by corresponding purchased detection kits, respectively. Cell apoptosis were measured by flow cytometry. Then binding effect of miR-208a on CHD9 3'UTR was detected by Dual-Luciferase activity assay. After miRNA or CHD9 overexpression transfections, expressions of apoptosis-related factors, CHD9, Notch 1, IκBα, and p65 in H9c2 cells after I/R injury were measured by Western blot assay. Results showed that in H9c2 cells after simulated I/R injury, miR-208a was upregulated. The elevated miR-208a expression enhanced the injury of cells and promoted cell apoptosis. miR-208a directly target 3'UTR of CHD9 and negatively regulated CHD9 expression. Overexpression of CHD9 rescued I/R injury that was enhanced by miR-208a mimic transfection. miR-208a was positively related with activation of Notch/NF-B signal pathways via CHD9. In conclusion, miR-208a was a cardiac-enriched miRNA and CHD9 is a direct target of miR-208a, which was also related with Notch/NFB signal pathway during I/R injury. miR-208a has potential to be a biomarker for early diagnosis of I/R injury and might be used as a treatment target in clinical treatment of ischemic heart disease.