Middle cerebral artery occlusion (MCAO) injury refers to impaired blood supply to the brain that is caused by a cerebrovascular disease, resulting in local brain tissue ischemia, hypoxic necrosis, and rapid neurological impairment. Nevertheless, the mechanisms involved are unclear, and pharmacological interventions are lacking. 25-Hydroxycholesterol (25-HC) was reported to be involved in cholesterol and lipid metabolism as an oxysterol molecule. This study aimed to determine whether 25-HC exerts a cerebral protective effect on MCAO injury and investigate its potential mechanism. 25-HC was administered prior to reperfusion in a mouse model of MCAO injury. 25-HC evidently decreased infarct size induced by MCAO and enhanced brain function. It reduced stimulator of interferon gene (STING) activity and regulated mTOR to inhibit autophagy and induce cerebral ischemia tolerance. Thus, 25-HC improved MCAO injury through the STING channel. As indicated in this preliminary study, 25-HC improved MCAO injury by inhibiting STING activity and autophagy as well as by reducing brain nerve cell apoptosis. Thus, it is a potential treatment drug for brain injury.