Abstract: Objective To observe the effect of autophagy-related protein 5（ATG5） on mitophagy in oxygen-glucose deprivation/reoxygenation（OGD/R）-induced H9c2 cardiomyocytes. Methods OGD/R was used to simulate myocardial ischemia-reperfusion injury in H9c2 cardiomyocytes. H9c2 cells were treated as follows: no treatment（control group）, OGD/R model（OGD/R group）, OGD/R+transfection of empty vector（OGD/R-Vehicle group）, OGD/R+ transfection of ATG5 overexpression plasmid（OGD/R-ATG5 group）, OGD/R+meaningless sequence interference RNA（OGD/R-siRNA-negative group） and OGD/R+silencing ATG5 gene sequence（OGD/R-siRNA-ATG5 group）. Cell counting kit 8（CCK8） was used to detect the proliferation of H9c2 cells. Mitochondrial survival was observed under microscope. Reverse transcription-quantitative polymerase chain reaction（RT-qPCR） and western-blot were used to detect the mRNA and protein expressions of ATG5, FUN14 domain-containing protein 1（FUNDC1）, dynamin-related protein 1（DRP1）, and optic atrophy associated protein 1（OPA1）. Western-blot was used to detect the expressions of microtubule-associated protein 1A/1B-light chain 3 Ⅱ（LC3Ⅱ） and nuclear porin glycoprotein P62（P62）. Results Compared with the control group, the proliferation rate of H9c2 cells in OGD/R group, OGD/R-Vehicle group, OGD/R-ATG5 group, OGD/R-siRNA negative group and OGD/R-siRNA-ATG5 group was decreased（F=106.35, P＜0.05）. Mitochondrial activity was decreased in OGD/R, OGD/R-vehicle, OGD/R-siRNA negative and OGD/R-siRNA-ATG5 groups, while increased in OGD/R-ATG5 group（F=50.74, P＜0.05）. The expression levels of ATG5, FUNDC1 and DRP1 mRNA and protein in OGD/R group, OGD/R-vehicle group, OGD/R-ATG5 group and OGD/R-siRNA negative group were increased, while the mRNA and protein expressions of ATG5, FUNDC1 and DRP1 were decreased in OGD/R-siRNA-ATG5 group（all P＜0.05）. The expression levels of OPA1 mRNA and protein in all groups were lower than those in the control group（F=9.924, 24.334, P＜0.05）. The expression level of LC3Ⅱ protein in OGD/R group, OGD/R-Vehicle group, OGD/R-ATG5 group and OGD/R-siRNA negative group was increased, and the expression level of P62 protein was decreased（F=18.07, 17.50, P＜0.05）. In the OGD/R-siRNA-ATG5 group, the expression level of LC3Ⅱ protein was decreased, and the expression level of P62 protein was increased. Compared with group OGD/R, the cell proliferation rate, mitochondrial activity, mRNA and protein expressions of ATG5, FUNDC1, DRP1 and OPA1 were significantly increased, the protein expression of LC3Ⅱ was increased, and the protein expression of P62 was decreased in group OGD/R-ATG5. In the OGD/R-siRNA-ATG5 group, the cell proliferation rate, mitochondrial activity, mRNA and protein expressions of ATG5, FUNDC1, and OPA1, DRP1 protein expression, LC3Ⅱ protein expression, and P62 protein expression were decreased（all P＜0.05）. There was no significant difference in all indexes between OGD/R-vehicle group and OGD/R group, OGD/R-siRNA negative group and OGD/R group（P＞0.05）. Conclusion H9c2 cells show mitochondrial fission and mitophagy after OGD/R injury, and ATG5 can enhance mitophagy to alleviate OGD/R injury in cardiomyocytes.