Myocardial ischemia–reperfusion (I/R) injury severely impacts the postoperative survival rate of coronary atherosclerotic heart disease. Radioprotective 105kDa protein (RP105) is a regulator of Toll-like receptor 4 (TLR4), an inflammatory factor whose functions have been reported in myocardial I/R injury. To investigate the roles of RP105 in mediating myocardial I/R injury, we overexpressed RP105 by injecting its adenovirus vectors, and induced myocardial I/R injury rat model in this study. Myocardial structure injuries of rat hearts were examined by hematoxylin eosin staining, and myocardial infarct area was calculated after Evans blue and triphenyltetrazolium chloride dual staining. Expression changes of TLR4, myeloid differentiation factor 88 (MyD88), and nuclear factor κB (NF-κB) in myocardia were detected by quantitative real-time PCR and Western blot. Amount changes of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay. Results showed that RP105 attenuated myocardial injuries and effectively reduced myocardial infarct area after I/R (P<0.05). RP105 was also proved to significantly inhibit TLR4 and downstream inflammatory factors MyD88, NF-κB, TNF-α and IL-6 (P<0.05), whose expression levels were up-regulated by I/R induction. These results indicated that RP105 could protect against myocardial I/R injury via suppressing inflammatory responses mediated by TLR4 signaling pathways. This study revealed the anti-inflammatory roles of RP105 and its potential in preventing and treating myocardial I/R injury.