Objective To investigate the effect of hydrogen peroxide (H2O2) pretreatment on free mitochondrial cyto-chrome c (Cyt-c) release in mitochondrial levels, and reveal the mechanism of the ischemic preconditioning (IPC) on the ischemia reperfusion (IR) injury thereof. Methods The rat liver mitochondria was isolated and made free mitochondria. Free mitochondria were divided into 5 groups:control group (C) and different concentrations of Ca2+groups (12.5, 25, 50 and 100 μmol/L). The levels of Cyt-c and second mitochondria-defived activator of caspase (Smac) were detected after 10 min stimulation of free mitochondria. The free mitochondrial IPC reperfusion model was made and divided into seven groups:C group, IR group and different concentrations of H2O2 groups (2 μL H2O2 in 200 μL system respectively, final concentration of 1, 2, 5, 20 and 100 μmol/L respectively). 100 μmol/L Ca2+was used again on the simulation of IR group. The level of Cyt-c release was detected. The changes in the activity of cardiolipin were detected in IR group and H2O2 (1 and 2 μmol/L of final concentration) groups.Results Compared with C group, there were significantly higher levels of Cyt-c and Smac emission in 25, 50, and 100 μmol/L Ca2+groups (P<0.05). Compared with IR group, there was significantly decreased level of Cyt-c emission in H2O2 (1 and 2 μmol/L) groups (P < 0.05). The activity of cardiolipin was changed when reducing release of Cyt-c. Conclusion Cyt-c was bonded with cardiolipin more closely when the low concentration of H2O2 pretreatment in mitochondria. There was a lower level of Cyt-c emission in mitochondria after stimulation with high concentration of Ca 2+(100 μmol/L Ca2+). The blocking apoptotic pathway plays a key fact in the effect of IPC on IR injury.