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Introduction: Hypothermia treatment can provide cardiac protection against ischemia reperfusion injuries, but underlying mechanisms remain unclear. Hypothesis: Hypothermia-related cardiomyocyte protection is through the mitochondrial dependent pathways. Methods : H9c2 rat cardiomyocytes were cultured in 37°C, 5% CO 2 incubators. After initiation of hypoxia-reoxygenation (H-R) treatment, the H9c2 cells were moved to hypothermia (31°C) or kept in normothermia (37°C) environments until cells harvested. Cell damage, intracellular and mitochondria calcium loads were studies. Mitochondria permeability transition and transmembrane potentials were studies by flowcytometric studies. Results: Hypothermia treatment ameliorates H9c2 cardiomyocytes survival after H-R injuries (68.1±11.8% vs. 85.0±12.7 %, P=0.025). Intracellular and mitochondria calcium overloading after H-R injuries was improved under 31°C environment (153.5±16.4 % vs. 957.1±311.7 %, P<0.01 for intracellular calcium; 101.8±28.5% vs. 159.4±32.5%, P=0.014 for mitochondria calcium). Mitochondria reductase activity were more preserved under hypothermia treatment after H-R injuries (55.7±10.9% vs. 8.5±1.2%, P<0.01). Hypothermia treatment decreased the continuous opening of mitochondria permeability transition pore after H-R damage by less reduction of mitochondria calcein fluorescence (15.6±13.7 % vs. 52.8±28.1 %, P=0.003). Release of cytochrome c into the cytoplasm from mitochondria after H-R injuries was more evident in normothermia condition by confocal microscopy study. Activation of caspase-9, which is down-streaming to cytochrome c, was down-regulated under hypothermia (62.1±21.9% vs. 87.5±7.3%, P=0.019). Loss of mitochondria integrity with decreasing of mitochondria transmembrane potential was less evident in 31°C than 37°C environments (55.9±23.3% vs. 102±20.2%, P=0.016). Conclusion: Hypothermia treatment at 31°C provides cardiomyocyte protection against hypoxia-reoxygenation injuries. The mechanisms are related to the decreasing intracellular and mitochondria calcium overloading, preserving the integrity of mitochondria by reduction of mitochondria permeability transition pore opening and cytochrome c release.