Abstract: The model of hypoxic injury of H9C2 cardiomyocytes was established with cobalt chloride (CoCl₂) to study the role of Nardosinone (Nar) in it and its mechanism. CCK-8 kit was used to detect cell viability and flow cytometry was used to detect apoptosis. Autophagy inhibitor 3-MA was used to pretreat cells to evaluate the effect of Nar on autophagy. The expressions of Beclin-1, LC3II/LC3I, P62, Bax, Bcl-2 and Caspase-3 were detected with Western Blot, and the contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA) and creatine kinase (CK) were measured with spectrophotometer. The results showed that CoCl₂(500 μmol/L) inhibited the growth of H9C2 cells by about 50% while the pretreatment with Nar(50 μmol/L) significantly alleviated CoCl₂-induced cell apoptosis. In addition, Nar promoted P62 degradation and increased the expressions of LC3II/LC3I and Beclin-1, which were reversed with 3-MA pretreatment. At the same time, pretreatment with 3-MA reversed the protective effects of Nar on CoCl₂-caused H9C2 cell apoptosis and oxidative damage. The results showed that Nar played a protective role in myocardial hypoxic injury by inducing autophagy and inhibiting apoptosis. It was suggested that Nar may be a potential drug for the treatment of hypoxic heart disease.