This study was designed to explore the role of toll like receptor 4 (TLR4) in myocardial ischemia reperfusion injury (MI/RI). Male Sprague-Dawley rats were randomly divided into sham and IR groups (36/group). The rats were sacrificed at various times following reperfusion (0, 1/2, 1, 2, 4 and 8 h). The histopathological and ultrastructural changes in the myocardium were examined under a light microscope and a transmission electron microscope. The TLR4 protein and mRNA expression were detected by immunohistochemistry and real-time reverse transcription polymerase chain reaction, respectively. The levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in the myocardium were measured by enzyme-linked immunosorbent assays. The injury to the myocardium was severe in the IR group, and there were no significant improvements in histopathology and ultrastructure of the myocardium during the first 8 h following reperfusion. Positive TLR4 protein staining was observed in both sham and IR groups. The TLR4 protein levels were significantly increased in the IR group, peaking at 1 h post reperfusion. Additionally, the TLR4 mRNA levels were also up-regulated in the IR group. At all time points, IR rats had significantly higher TNF-alpha and IL-6 levels than the sham rats (P<0.05). The TLR4 mRNA expression positively correlated with the levels of TNF-alpha and IL-6 (r=0.728 and 0.676, P<0.01). Myocardial TLR4 expression was elevated at the early stage of myocardial ischemia reperfusion. Activated TLR4 may play a role in MI/RI by increasing TNF-alpha and IL-6 expression.