Eupatorium perfoliatum L. (E. perfoliatium) has been used traditionally for treating fever, malaria and inflammation‑associated diseases. Eupafolin, the extract of E. perfoliatium, was also reported to suppress inflammation. The present study aimed to investigate the protective effects of eupafolin on cerebral ischemia/reperfusion (I/R) injury in rats and its possible underlying mechanisms. Cerebral I/R injury was induced in rats by middle cerebral artery occlusion (MCAO) for 1.5 h, followed by reperfusion. The rats were randomly assigned into six groups: Control, model, 10 mg/kg eupafolin, 20 mg/kg eupafolin, 50 mg/kg eupafolin and 20 mg/kg nimodipine. Eupafolin and nimodipine were intragastrically administrated to the rats 1 week before MCAO induction. Following reperfusion for 24 h, the neurological deficit was scored, and brain samples were harvested for evaluating encephaledema, infarct volume, oxidative stress, apoptosis, inflammation and the expression of TLR4/NF‑κB signaling. The results revealed that eupafolin decreased the neurological score, relieved encephaledema and decreased infarct volume. Eupafolin also attenuated oxidative stress, neuronal apoptosis and inflammation, with decreases in lactate dehydrogenase, malondialdehyde, TUNEL‑positive cells, Bax and caspase‑3, along with TNF‑α, IL‑1β and IL‑6, but increases in superoxide dismutase and Bcl‑2 levels. Furthermore, eupafolin may decrease the expression of TLR4 downstream proteins and proteins involved in the NF‑κB pathway. Treatment with TLR4 agonist‑LPS significantly blunted the protective effect of eupafolin on encephaledema and cerebral infarct. Meanwhile, 20 mg/kg eupafolin showed nearly equivalent effects to the positive‑control drug nimodipine. In conclusion, eupafolin protected against cerebral I/R injury in rats and the underlying mechanism may be associated with the suppression of apoptosis and inflammation via inhibiting the TLR4/ NF‑κB signaling pathway.