Both of gp91(phox) (an isoform of nicotinamide adenine dinucleotide phosphate reduced oxidases) and Src (a nonreceptor protein tyrosine kinase) are abundantly expressed in the brain and play a prominent role in mediating ischemic alteration in neurons. The inhibitory strategy of them is believed to be the promising treatment of stroke. The present study was designed to investigate the effect of equol (0.625-2.5 mg·kg(-1), i.g. for 3 days), a predominant active metabolite of daidzein, on neuroprotection against cerebral ischemia/reperfusion injury in rats and the underlying mechanisms. We found that equol decreased the mortality, neurological deficit, brain histological damage, infarct volume, serum lactate dehydrogenase activity and malondialdehyde content in a dose-dependent manner in rats with 2-h middle cerebral artery occlusion, followed by 22-h reperfusion. Western blot analysis revealed that protein levels of gp91(phox) and phosphorylated Src-Tyr416 (p-Src) in ischemic cerebral cortex were increased in rats treated with vehicle, which was reversed in animals treated with equol. In rat pheochromocytoma cell line (PC12) with hypoxia/reoxygenation injury, silencing of gp91(phox) with specific siRNA did not affect the increase of p-Src level by hypoxia/reoxygenation injury and the inhibition of p-Src level by equol, while silencing of Src suppressed the upregulation of gp91(phox) by hypoxia/reoxygenation injury and enhanced the inhibitory effect of equol on gp91(phox) expression. These results demonstrate that equol confers a neuroprotection in rats via inhibiting the activation of Src and upregulation of gp91(phox) induced by focal cerebral ischemia/reperfusion, and Src may play a partial role in regulating gp91(phox) expression of neurons.