Objective To investigate the effects of isoflurane preconditioning on the expression of p38 mitogen-activated protein kinase (p38 MAPK) in myocardium in a rat model of myocardial ischemia-reperfusion (I/R). Methods Ninety male Wistar rats weighing 270-390 g were anesthetized with intraperitoneal thiobutabarbital 150 mg/kg, tracheostomized and mechanically ventilated. I/R were produced by reversible occlusion of left anterior descending branch (LAD) of coronary artery for 30 min followed by 2-hour reperfusion. Then, sixty animals were randomly divided into 6 groups (n=10 for each): (1) control group received intravenous normal saline (NS) before I/R; (2) SB group received SB203580 (p38 MAPK inhibitor) 1.5 mg/kg iv before I/R; (3) DMSO group received DMSO (the SB203580 solvent) 0.2 mL before I/R; (4) ISO group inhaled 1.0 MAC isoflurane for 30 min followed by 15 min wash-out before I/R; (5) SB+ISO group and (6) ISO+SB group received SB 1.5 mg/kg before and after 30 min 1.0 MAC isoflurane inhalation. At the end of 2-hour reperfusion, the size of myocardial infarct was determined using triphenyl tetrazolium chloride staining. Another 30 animals were randomly divided into 5 groups (n=6 for each): control group, SB group, ISO group, SB+ISO group and ISO+SB group. Before occlusion of LAD and at the end of 2-hour reperfusion, myocardial specimens were obtained for determination of expression of p38 MAPK protein in myocardium (Western blot). Results SB group, ISO group, SB+ISO group and ISO+SB group significantly reduced infarct size. The expression of p38 MAPK protein was up-regulated in CON group and ISO group after 2-hour reperfusion as compared to the CON group before occlusion of LAD. Conclusions Isoflurane preconditioning and p38 MAPK inhibitor SB203580 can protect myocardium against I/R injury, but p38 MAPK may not be involved in the mechanism of isoflurane preconditioning.