Aim To explore the role of c-Jun N-terminal kinase （JNK） pathway in high glucose-induced cardiomyocyte injury and whether hydrogen sulfide （H₂S） protects cardiomyocytes against high glucose-induced injury by modulating JNK pathway. Methods H9c2 cells were treated with high glucose to establish a model of cardiac cell injury. CCK-8 was used to detect cell viability, intracellular level of reactive oxygen species （ROS） was measured by DCFH-DA staining and photofluorography, mitochondrial membrane potential （MMP） was tested by Rh123 staining and photofluorography, the activation of JNK protein was measured by Western blot assay. Results Glucose at high concentration induced obvious injury of H9c2 cells, leading to a decrease in cell viability, an increase in ROS generation and MMP loss, as well as upregulation of phosphorylated JNK expression Pretreatment with N-acethl-L-cystein （NAC）, a ROS scavenger or SP600125 （an inhibitor of JNK） for 30 min inhibited high glucose-induced cytotoxicity Pretreatment with 400 μmol/L NaHS （a donor of H₂S） for 30 min prior to exposure of H9c2 cells to high glucose blocked high glucose-induced injuries, evidenced by an increase in cell viability, decreases in ROS generation and loss of MMP, along with inhibition of phosphorylated JNK expression induced by high glucose. Conclusion Both ROS and JNK pathway mediate H9c2 cardiac cell injury induced by high glucose, H₂S may protect cardiac cells against high glucose-induced injury by inhibiting oxidative stress and JNK pathway.