Aim To investigate the roles of the interaction between reactive oxygen species （ROS） and ATP-sensitive K^＋ （K_ATP） channel in high glucose （HG）-induced cardiac muscle cells injury.Methods The expression levels of K_ATP channel protein and cleaved caspase-3 protein were determined by Western blot assay. The intracellular level of ROS was detected by 2',7'-dichlorfluorescein-diacetate （DCFH-DA） staining and fluorescence microscopy. The cell viability was measured by cell counter kit-8 （CCK-8） assay. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining and photofluorography. Mitochondrial membrane potential （MMP） was examined by JC-1 staining.Results After H9c2 cells were treated with 35 mmol/L glucose （high glucose, HG） for 24 h, the expression level of K_ATP channel protein was significantly reduced. Pretreatment of the cells with 1000 μmol/L N-acetyl-L cysteine （NAC, a scavenger of ROS） for 60 min before HG exposure obviously reduced the HG-induced inhibitory effect on the expression of K_ATP channel protein. Meanwhile, pretreatment of the cells with 100 μmol/L diazoxide （DZ, a mitochondrial K_ATP channel opener） or 50 μmol/L pinacidil （Pin, a none-selective K_ATP channel opener） markedly inhibited HG-induced accumulation of intracellular ROS in cardiac cells. On the other hand, 1000 μmol/L NAC, 100 μmol/L DZ and 50 μmol/L Pin obviously inhibited HG-induced cardiac muscle cells injury, leading to an increase in cell viability, a decrease in number of apoptotic cells, cleaved caspase-3 expression and MMP loss respectively.Conclusion In HG state, there is an interaction between ROS and K_ATP channel, which plays an important role in the HG-induced injury in cardiac muscle cells.