Abstract Background Our previous studies indicate that either PEP-1-superoxide dismutase 1 (SOD1) or PEP-1-catalase (CAT) fusion proteins protects myocardium from ischemia-reperfusion-induced injury in rats. The aim of this study is to explore whether combined use of PEP-1-SOD1 and PEP-1-CAT enhances their protective effects. Methods SOD1, PEP-1-SOD1, CAT or PEP-1-CAT fusion proteins were prepared and purified by genetic engineering. In vitro and in vivo effects of these proteins on cell apoptosis and the protection of myocardium after ischemia-reperfusion injury were measured. Embryo cardiac myocyte H9c2 cells were used for the in vitro studies. In vitro cellular injury was determined by the expression of lactate dehydrogenase (LDH). Cell apoptosis was quantitatively assessed with Annexin V and PI double staining by Flow cytometry. In vivo, rat left anterior descending coronary artery (LAD) was ligated for one hour followed by two hours of reperfusion. Hemodynamics was then measured. Myocardial infarct size was evaluated by TTC staining. Serum levels of myocardial markers, creatine kinase-MB (CK-MB) and cTnT were quantified by ELISA. Bcl-2 and Bax expression in left ventricle myocardium were analyzed by western blot. Results In vitro, PEP-1-SOD1 or PEP-1-CAT inhibited LDH release and apoptosis rate of H9c2 cells. Combined transduction of PEP-1-SOD1 and PEP-1-CAT, however, further reduced the LDH level and apoptosis rate. In vivo, combined usage of PEP-1-SOD1 and PEP-1-CAT produced a greater effect than individual proteins on the reduction of CK-MB, cTnT, apoptosis rate, lipoxidation end product malondialdehyde, and the infarct size of myocardium. Functionally, the combination of these two proteins further increased left ventricle systolic pressure, but decreased left ventricle end-diastolic pressure. Conclusion This study provided a basis for the treatment or prevention of myocardial ischemia-reperfusion injury with the combined usage of PEP-1-SOD1 and PEP-1-CAT fusion proteins.