Recent studies have demonstrated nuclear factor-κB (NF-κB) and high-mobility group box1 (HMGB1) associated with the pathophysiology of cerebral ischemia. We isolated Curculigoside A, the major bioactive compound present in Curculigo orchioides. The objectives of this study were to determine the effects of Curculigoside A on cultured neuronal cell line, SH-SY5Y in vitro and experimental ischemic stroke in vivo. For oxygen-glucose deprivation and tumor necrosis factor-α (TNF-α) stimulated SH-SY5Y cell line in vitro, SH-SY5Y cells were pre-incubated with Curculigoside A. For in vivo experiment, rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h, then followed by reperfusion for 23 h. Treatment of SH-SY5Y cells with Curculigoside A reduced the oxygen-glucose deprivation-induced cytotoxicity and apoptosis, blocked TNF-α-induced NF-κB and IκB-α phosphorylation, and decreased HMGB1 expression. At doses higher than 10 mg/kg, Curculigoside A produced a significant neuroprotective potential in rats with ischemia and reperfusion (I/R). Curculigoside A (20 mg/kg) demonstrated significant neuroprotective activity even after delayed administration at 1 h, 3 h, and 5 h after I/R. Curculigoside A 20 mg/kg attenuated histopathological damage, decreased cerebral Evans Blue extravasation, inhibited NF-κB activation and reduced HMGB1 expression. These data show that Curculigoside A protects brain against I/R injury with a favorable therapeutic time-window by alleviating cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and its protective effects may involve HMGB1 and NF-κB signaling pathway.