Objective To investigate the effect of mild hypothermia therapy after CPR on the expression of NF-κB and HIF-1α in rat′s myocardial cells. Methods Twenty SD rats were made the mode of CPR by clamping the trachea. The rats were randomly divided into four groups: Sham operation group, room temperature treated group, 34 ℃ treated group, 32 ℃ treated group. The sham operation group was treated by catheterization of arterial and venous, tracheal intubation, and the myocardial tissue samples were collected 6 hours later. For the other groups, the myocardial tissue samples were collected 6 hours after CPR. Western-blotting was used to test HIF-1α and NF-κB protein expression of each group′s myocardial tissue. The optical microscopy was used to observe the injury of myocardial tissue. Results The expression of HIF-1α and NF-κB protein in room temperature treated group, 34 ℃ treated group and 32 ℃ treated group was significantly higher than that in sham operation group(P＜0.01). The expression of HIF-1α and NF-κB protein in 34 ℃ treated group and 32 ℃ treated group was significantly lower than room temperature treated group(P＜0.05). The expression of NF-κB protein in 32 ℃ treated group was significantly lower than that in 34 ℃ treated group(P＜0.05), but the expression of HIF-1α protein in 32 ℃ treated group was not significantly lower than that in 34 ℃ treated group(P＞0.05). Conclusion Mild Hypothermia therapy can protect the myocardial cells after CPR through decreasing the expression of HIF-1α and NF-κB protein. The hypothermia of 32 ℃ plays a better protective role in the injury of reperfusion than 34℃, but it increases the side effect such as amyostasia, which should be taken seriously.