Objective：This study aims to detect the effect of mitochondria from high fat diet mice heart，and the effects of hypoxia-reoxygenation. Methods：A total of 12 2-week-old mice（male，C57B6）were randomly divided into high-fat-diet（HFD，n=6）group and control（CTR，n=6）group，and they were fed with high fat diet and normal diet respectively for 20 weeks. Hearts were used to isolate pure mitochondria. After the oxygen used up in the chamber，the mitochondria were subjected to 30 minutes of hypoxia-reoxygenation to simulate ischemia-reperfusion. Mitochondrial oxygen consumption rate（OCR）was measured using oxygen monitor system. Mitochondrial complex enzyme activity was assessed using microplate colorimetric assay kit. Reactive oxygen species（ROS）was measured by fluorimeter. Results：The mitochondrial OCR was greater in HFD group compared to that of CTR group（P < 0.05）. Similarly，mito-complex Ⅱ activity was significantly increased in HFD group compared to that of CTR group（P < 0.05）. Furthermore，reoxygenation of purified mitochondria following 30 min hypoxia transiently increased OCR，with significantly higher increase in HFD group. Pre-treatment of mito-complex Ⅱ inhibitor，malonate diminished reoxygenation-induced OCR increase in both groups. The similar tendency was also detected in ROS. Conclusion：Mito-complex Ⅱ activity was totally enhanced in the HFD model，which could be involved in the injury of hypoxia-reoxygenation（which simulated ischemia-reperfusion）in heart.