bjective To explore the possible mechanism by which the antioxidant N‑acetylcysteine (NAC) reduces hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes and the role of mitophagy in the process. Methods H9c2 cardiomyocytes were cultured in vitro. According to the random number table method, they were divided into three groups (each group had three replicate wells): a control (NC) group, a hypoxia/reoxygenation (H/R) group, and a hypoxia/reoxygenation+N‑acetylcysteine (H/R+NAC) group. Cells in the NC group were normally cultured. Those in the H/R group were subjected to hypoxia for 4 h, followed by reoxygenation for 4 h. The H/R+NAC group were subjected to hypoxia for 4 h, followed by reoxygenation for 4 h, with the presence of NAC at a final concentration of 1.5 mmol/L. The cell counting kit‑8 (CCK‑8) was used to detect cell viability, while the enzyme‑linked immunosorbent assay (ELISA) was used to detect lactate dehydrogenase (LDH) levels in culture media. In addition, the 2, 7‑dichlorodihydrofluorescein ‑diacetate (DCFH‑DA) fluorescent probe method was used to measure the levels of intracellular reactive oxygen species (ROS). The 5, 5', 6, 6'‑tetrachloro‑1, 1', 3, 3'‑tetraethylbenzimidazolecarbocyanine iodide (JC‑1) method was used to monitor the levels of mitochondrial membrane potential. Western blot was used to detect the protein levels of mitophagy molecule proteins, P62, microtubule‑associated protein 1 light chain3Ⅱ (LC3Ⅱ), PTEN‑induced putative kinase 1 (PINK1), and Parkinson's disease protein (Parkin). Flow cytometry was conducted to estimate apoptotic levels. Results Compared with the NC group, the H/R group showed reduced cell survival rate (P<0.05), increased LDH and ROS levels (all P<0.05), reduced levels of mitochondrial membrane potential and P62, LC3Ⅱ, PINK1, and Parkin proteins (all P<0.05), with an increased cell apoptosis rate (P<0.05). Compared with the H/R group, the H/R+NAC group exhibited increased cell survival rate (P<0.05), decreased LDH and ROS levels (all P<0.05), increased levels of mitochondrial membrane potential and P62, LC3Ⅱ, PINK1, Parkin proteins (all P<0.05), with a decreased cell apoptosis rate (P<0.05). There was no statistical difference in cell survival rate, LDH, ROS, mitochondrial membrane potential levels, the levels of P62, LC3Ⅱ, PINK1 and Parkin protein, and cell apoptosis rate between the H/R+NAC group and the NC group (all P>0.05). Conclusion NAC can alleviate the damage of H9c2 cells caused by H/R through promoting mitophagy.