BACKGROUND Methylprednisolone has been demonstrated to decrease inflammation, and it may protect organs from ischemia/reperfusion (I/R) injury. This study aimed to investigate the effects of methylprednisolone on diabetic myocardial I/R injury. METHODS Forty adult male Sprague-Dawley (SD) rats were randomized into five groups (n = 8 in each group) including a sham operation (sham) group, I/R group, diabetic sham operation (DMS) group, diabetic I/R (DM-I/R) group and methylprednisolone intervention (MP + DM-I/R) group. The diabetic model was produced by injection of streptozotocin (STZ). Body weight and blood glucose levels were determined after diabetes was established. Twelve weeks after induction of diabetes, a segmental I/R of the heart was induced by occluding the left anterior descending artery for one hour and then three hours of reperfusion in the I/R, DM-I/R and MP + DM-I/R groups. Blood pressure and electrocardiogram were continuously recorded during the procedure. IL-1β, IL-6 and TNF-α were measured at certain time points during the surgery. After reperfusion, a microcirculation scan was performed; myocardial biomarkers and tissue structure were utilized to evaluate the reperfusion damage. Intercellular adhesion molecule (ICAM)-1 and NF-κBp65 expression were quantified by immunohistological staining. Total Toll-like receptor 4 (TLR4) and nuclear NF-κBp65 protein were determined by Western blotting. RESULTS Twelve weeks after diabetes was established, blood glucose levels were elevated and body weights were lower in diabetic rats. After reperfusion, infarction size was increased, myocardial biomarkers and inflammatory cytokines levels were elevated. Microcirculation perfusion was significantly reduced in the DM-I/R group compared with the I/R group, however it was improved in the MP + DM-I/R group. The expression of NF-κBp65 and ICAM-1 were increased in the DM-I/R group and decreased in the MP + DM-I/R group. Compared with the non-diabetic I/R group, TLR4 and NF-κBp65 protein levels were up-regulated in the DM-I/R group, but down-regulated in the MP + DM-I/R group. CONCLUSIONS Methylprednisolone improves microcirculation in STZ-induced diabetic rats after myocardial ischemia/reperfusion, which may associate with the suppression of TLR4/NF-κB signaling.