Objective: To detect the change of mitochondrial function in the early stage after myocardial ischemia-reperfusion injury in mice. Methods: C57BL/6 mice were divided randomly to five groups. Hearts in the time control group (TC) were perfused for 45 min in identical Krebs-Henseleit buffer without any treatment. In ischemia-reperfusion groups, hearts were given different reperfusion time including 5 min, 10 min, 15 min or 30 min followed the same ischemia period 25 min. Mitochondria were extracted from left ventricular after reperfusion. Then respiratory function, mitochondrial calcium retention capacity and inner mitochondrial membrane integrity were measured. Results: The results revealed that R3, RCR and P/O of mitochondria were decreased significantly after ischemia and reperfusion 10 min, 15 min and 30 min (P<0.05), but not in reperfusion 5 min group compared with time control group using glutamate + malate as substrates. And in different ischemia-reperfusion groups respiratory function decreased significantly compared with time control group using succinate, and TMPD-ascorbate as substrates (P<0.05). Meanwhile, both mitochondrial calcium retention capacity and inner mitochondrial membrane potential decreased gradually following prolonged reperfusion. Conclusions: Mitochondrial respiratory function changes differently in different complex at early stage after ischemia and reperfusion. So we should choose different ischemiareperfusion time to detect different mitochondrial complex changes after heart injury.