Objective：To research the effect of methylene blue（MB） pretreatment on focal cerebral ischemia-reperfusion（I/R） injury in rats and its possibly mechanism. Methods：Sixty-four adult male Sprague-Dawley rats were randomly divided into four groups（n=16）：sham group（group S），methylene blue control group （group S+M），focal cerebral ischemia-reperfusion group（group I/R），methy－lene blue pretreatment group （group I/R+M）. The rat model of focal cerebral ischemia-reperfusion was established by middle cerebral artery occlusion. The neurological deficit scores were performed with Longa’s scale at 5 min before reperfusion and at 24 h after reperfusion. The pathological changes in pallium of ischemic area were observed by HE staining. Reactive oxygen species（ROS） in ischemic brain tissue were measured by chemiluminescent. The expressions of nuclear factor erythroid 2-related factor 2（Nrf2） and heme oxygenase 1（HO-1） in ischemic brain tissue were detected by Western blot and immunohistochemistry staining. Results：The neurological deficit scores，the cerebral infarct volume and the infarct volume fraction，the content of ROS，the expression of Nrf2 and HO-1 were all obviously higher in group I/R and I/R+M than in group S and S+M respectively（P<0.01）. The neurological deficit scores，the cerebral infarct volume and the infarct volume fraction，the content of ROS were all obviously lower in group I/R+M than in group I/R（P<0.01）. The expressions of Nrf2 and HO-1 were obviously increased in group I/R+M than in group I/R（P<0.01）. The pathological changes in pallium of ischemic area were significantly ameliorated in group I/R+M than in group I/R. The difference between group S and group S+M was not statistical significant（P>0.05）. Conclusion：The pretreatment of methylene blue can improve the focal cerebral ischemia-reperfusion injury in rats，which might be related to upregulated the expression of Nrf2 and HO-1，reduce the content of ROS in brain，produce antiox－idant stress.