Autophagy plays a critical role in cardiac hypoxia/reoxygenation (H/R). Studies indicated that the phosphatase and tensin homolog (PTEN) influences level of autophagy. This study aims to explore the role of PTEN mediating a specific autophagy, mitophagy, in cardiac H/R injury. H9c2 cells were cultured and suffered hypoxia and reoxygenation treatment. To inhibit function of PTEN protein, bpv (phen) was added into medium throughout the process of H/R injury. In addition, we overexpressed the apurinic/apyrimidinic endonuclease 1 (APE1) in H/R-injured H9c2 cells. Then the cell viability, apoptosis, and release of Cytochrome C were determined through CCK-8 assay, flow cytometry, and western blotting, respectively. The results indicated that H/R significantly induced autophagy, as identified by an increased level of microtubule-associated protein 1 light chain 3 beta (LC3B) and a decreased level of sequestosome 1 (P62). After stimulation of bpv (phen), PTEN-induced putative kinase protein 1 (PINK1)/Parkin-mediated mitophagy was inhibited, while apoptosis and releases of Cytochrome C were both significantly increased, indicating an exacerbated H/R injury. Furthermore, the overexpression of APE1 attenuated the apoptosis and releases of Cytochrome C induced by H/R injury, and promoted PINK1/Parkin-mediated mitophagy. Our findings provide an insight into the PTEN and APE1 overexpression protects against cardiac hypoxia/reoxygenation injury, which may be through inducing the PINK1/Parkin-mediated mitophagy.