Stroke is a leading cause of disability and death in the worldwide. Therefore, prevention of stroke is critically important. Genistein, a natural phytoestrogen extracted from soybeans, has been found to be a potential neuroprotective agent for stroke prevention. However, the role of genistein and its underlying mechanism in ovariectomized rats has been rarely evaluated. In this study, ovariectomized rats were treated with genistein (10 mg/kg) or vehicle daily for two weeks before they received middle cerebral artery occlusion (MCAO) and reperfusion. Seventy-two hours after reperfusion, the neurological function was evaluated by Garcia test, infarct volumes were detected by 2,3,5-triphenyltetrazolium chloride staining; and neuronal damage and cell apoptosis were detected by Nissl and Tunel staining in the ischemic penumbra, respectively. In addition, Western blotting was used to detect the activity of PI3K-Akt-mTOR signal pathway in the ischemic penumbra in different groups. And we found that genistein treatment in ovariectomized rats significantly improved neurological outcomes, reduced infarct volumes, decreased neuronal damage and cell apoptosis, and increased the activity of PI3K-Akt-mTOR signal pathway. Our findings indicated that treatment genistein could alleviate neuronal apoptosis induced by cerebral ischemia in ovariectomized rats via promoting the activity of PI3K-Akt-mTOR signal pathway, which provides a new molecular mechanism for the neuroprotective effects of genistein against stroke.