Objective To investigate the value of glucose and serum deprivation under hypoxia（1% O₂）（GSDH） treatment and H₂O₂ treatment in establishing a model of oxidative injury in H9C2 cardiomyocytes. Methods H9C2 cardiomyocytes were cultured，and when the cardiomyocytes were in good growth conditions，they were treated with the combination of low oxygen（1% O₂），low glucose（1.0 g/L），and serum deprivation or H₂O₂ 200 μmol/L alone for 24 hours. CCK8 assay was sued to measure the proliferation ability of cells；the apoptosis reagent（AnnexinV-FITC/PI） and Hoechst staining were used to measure cell apoptosis；reactive oxygen species（ROS） was used to measure the level of oxidative stress；BODIPY testing was used to measure the level of lipid peroxidation in cells；periodic acid-Schiff staining was used to measure the ability for glycogen synthesis；mitochondrial membrane potential assay kit with JC-1 staining was used to measure mitochondrial membrane potential；Western blot was used to measure the protein expression levels of the energy metabolism-related molecule AMP-activated protein kinase（AMPK） and the oxidative stress-related molecules NAD（P）H quinone dehydrogenase 1（NQO-1） and heme oxygenase-1（HO-1）. Results Compared with the blank group，both the GSDH treatment group and the H₂O₂ treatment group had a significant reduction in the viability of cardiomyocytes（P<0.05）. Compared with the blank group，both GSDH treatment and H₂O₂ treatment increased the levels of cardiomyocyte apoptosis，ROS and lipid droplet accumulation，and glycogen consumption，with a reduction in mitochondrial membrane potential in cardiomyocytes. However，compared with the H₂O₂ treatment group，the GSDH treatment group showed significantly greater increases in glycogen consumption and lipid droplet accumulation and a significant reduction in AMPK phosphorylation. Conclusion Both GSDH treatment and H₂O₂ treatment can cause oxidative injury to H9C2 cardiomyocytes，but compared with H₂O₂ treatment，the effect of GSDH treatment is more in line with the energy metabolism transition during myocardial injury in vivo，and therefore，it is expected to be used as a simpler and more convenient model of oxidative injury to cardiac myocardium for scientific research.