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Unlabelled: In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H(2)O(2)), IEC-6 cells were subjected to 20 μmol/L H(2)O(2) and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes., Results: Significant morphology damage was caused by exposure to H(2)O(2), and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H(2)O(2) treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H(2)O(2). Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H(2)O(2) group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H(2)O(2) induced IEC-6 cell apoptosis.