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Ischemic stroke leads to hypoxia-induced neuronal death and behavioral abnormity, and is a major cause of death in the modern society. However, the treatments of this disease are limited. Brilliant Blue FCF (BBF) is an edible pigment used in the food industry that with multiple aromatic rings and sulfonic acid groups in its structure. BBF and its derivatives were proved to cross the blood-brain barrier and have advantages on the therapy of neuropsychiatric diseases. In this study, BBF, but not its derivatives, significantly ameliorated chemical hypoxia-induced cell death in HT22 hippocampal neuronal cell line. Moreover, protective effects of BBF were attributed to the inhibition of the extracellular regulated protein kinase (ERK) and glycogen synthase kinase-3β (GSK3β) pathways as evidenced by Western blotting analysis and specific inhibitors. Furthermore, BBF significantly reduced neurological and behavioral abnormity, and decreased brain infarct volume and cerebral edema induced by middle cerebral artery occlusion/reperfusion (MCAO) in rats. MCAO-induced increase of p-ERK in ischemic penumbra was reduced by BBF in rats. These results suggested that BBF prevented chemical hypoxia-induced otoxicity and MCAO-induced behavioral abnormity via the inhibition of the ERK and GSK3β pathways, indicating the potential use of BBF for treating ischemic stroke