Objective To investigate the protective effect of NF-κB signaling pathway on myocardial ischemia-reperfusion injury model rats and the regulatory mechanism of mitochondrial function and myocardial energy metabolism. Methods 40 SD rats were divided into sham surgery (Sham) group, myocardial ischemia-reperfusion injury model (Model) group, NF-κB signaling pathway activator (LPS) group, and NF-κB signaling pathway inhibitor (SN50) group, with 10 rats in each group. After 3 days of tail vein administration, Sham group rats underwent sham surgery, while the other 3 groups established rat models of myocardial ischemia-reperfusion injury. The rat cardiac function, the Serum levels of creatine kinase isoenzyme (CK-MB), myoglobin (Mb), the cardiac troponin I (cTn I), the lactate dehydrogenase (LDH), superoxide dismutase (SOD), and ATP content in myocardial tissue were evaluated. The mitochondrial structure and changes in mitochondrial membrane potential of myocardial cells were observed and the myocardial infarction area and the level of myocardial tissue apoptosis and pathological changes in each group were detected. Results Compared with the model group, NF-κB signaling pathway activator (LPS) could reduce left ventricular short axis shortening rate (LVFS) and left ventricular ejection fraction (LVEF) in rats (P＜0.05), and increase left ventricular end diastolic diameter (LVDd) and left ventricular end systolic diameter (LVDs) (P＜0.05). LPS could elevated levels of CK-MB, Mb, cTn I, LDH, ROS, and SOD in serum (P＜0.05), cause mitochondrial membrane damage, increase myocardial tissue mitochondrial membrane potential and reducing ATP content (P＜0.05), increase myocardial infarction area, myocardial cell apoptosis level, aggravate myocardial injury, and myocardial fibrosis (P＜0.05), inhibit UCP1, ANT, ATP5a levels and promote mitochondrial cytochrome C protein release. NF-κB signaling pathway inhibitor (SN50) could increase LVFS, LVEF (P＜0.05), reduce LVDd, LVDs (P＜0.05), reduce the levels of CK-MB, Mb, cTn I, LDH, ROS, and SOD in serum (P＜0.05), improve mitochondrial membrane damage, reduce mitochondrial membrane potential in myocardial tissue, and increase ATP content in myocardial tissue (P＜0.05), reduce myocardial infarction area, decrease myocardial cell apoptosis level, improve myocardial injury, and inhibit myocardial fibrosis (P＜0.05), enhance the expression levels of UCP1, ANT, and ATP5a, and inhibit the release of mitochondrial cytochrome C protein. Conclusion Inhibiting the NF-κB signaling pathway can significantly improve myocardial injury in rats with myocardial ischemia-reperfusion injury model, enhance mitochondrial function, promote energy metabolism of myocardial cells, and provide ideas for clinical treatment of myocardial injury patients