Abstract: Background and Objective: Acute cerebral ischemia was caused by cerebral artery blockage, which was the most common type of cerebral ischemia and the second leading fatal disease. The aim of the current study was to analyze the effects of picroside II on brain tissues in rats following middle cerebral artery occlusion/reperfusion (MCAO/R). Materials and Methods: Healthy adult male Wistar rats were used to establish MCAO/R models by intraluminal thread methods. The experimental rats were randomly divided into sham, model, picroside II (Picr) and PET-α (Pifithrin-α hydrobromide, an inhibitor of p53) groups. The neurological deficit and infarct volume were assessed using mNSS test and TTC staining. The morphology and structure of cortical brain tissues were observed by H and E and transmission electron microscopy (TEM). Apoptosis was counted by TUNEL. Mitochondrial permeability transition pore (mPTP) was determined using spectrophotometer. The expressions of p-p53, Bcl-2, Bax, Cyt c and Caspase-3 were tested using WB. Results: Picroside II ameliorated the neurological dysfunction of MCAO/R rats, reduced the cerebral infarct volume and apoptosis accompanied by mPTP closing and the inhibition of p53 signaling pathway and PET-α also simulated the therapeutic effect by inhibiting p53 signaling pathway. Conclusion: It was concluded that the MCAO/R could activate the opening of mPTP and neuronal apoptosis by p53 signaling pathway in rats. Picroside II played a neuroprotective role in inhibiting the activation of p53 signaling pathway.