Objective: Abnormal p38 MAPK activation involves in ischemia-reperfusion (IR) injury. Isoflurane (ISO) is a clinically used inhaled anesthetic and protects myocardial I-R injury. Our study assessed whether ISO exerts a protective role in myocardial I-R injury. Methods: Rat myocardial I-R injury model was set followed by analysis of p-p38 MAPK expression in myocardial tissue by western blot, caspase-3 activity, as well as MDA and SOD content. Rats were assigned into Sham group, IR group, I-R + ISO group, followed by measuring p-p38 MAPK expression, caspase-3 activity, MDA and SOD, cell apoptosis and ROS content. Results: Compared with Sham group, MDA content, caspase-3 activity and p-p38 MAPK protein expression as well as ROS content and apoptosis rate in I-R model rats were significantly increased and SOD activity was significantly decreased. ISO pretreatment significantly reduced MDA content, caspase-3 activity, ROS content and apoptosis rate in I-R model rats, increased SOD activity and reduced p-p38 MAPK expression. Conclusion: Activation of p38MAPK signaling plays a role in mediating myocardial IR injury and cardiomyocyte apoptosis. ISO pretreatment inhibits oxidative stress and cardiomyocyte apoptosis and protects myocardial IR injury via inhibiting p38MAPK signaling.