The revascularization of blood vessels after myocardial infarction can lead to serious myocardial damage. Previous studies showed that radioprotective 105 kDa protein (RP105) is a specific negative regulator of myocardial ischemia reperfusion injury (MIRI). RP105 can modulate the Toll‑like receptor (TLR) 2/TLR4 signaling pathways. However, the synergistic effect of TLR2/4 regulated by RP105 during MIRI requires further investigation. To determine this effect, a MIRI model was established in rats in the present study. The expression of RP105 was depleted by transfecting RP105‑siRNA and then detected using western blotting. Furthermore, the myocardium tissue was stained with the hematoxylin and eosin staining. Knockdown of RP105 promoted the activity of serum myocardial enzymes during MIRI and increased myocardial infarction. The present results indicated that knockdown of RP105 activated the TLR2/4 signaling pathway by modulating the myeloid differentiation primary response 88 and NF‑κB signaling pathways. Furthermore, decreased expression of RP105 promoted myocardial cell apoptosis, which induced the damage of myocardial ischemic reperfusion. The present results suggested both TLR2 and TLR4 as key targets of RP105, thus RP105 may be a promising candidate to facilitate the development of novel therapeutic strategies for MIRI.