The phenolic glucoside gastrodin is the main component extracted from the rhizome of Gastrodia elata (Orchidaceae), a Chinese herbal medicine, which has long been used for treating dizziness, epilepsy, stroke and dementia. The present study aims to investigate the effect of gastrodin on hypoxia-induced neurotoxicity in cultured rat cortical neurons. Neuron survival and extracellular glutamate level were measured after an insult by hypoxia. Glutamate concentrations were determined by an HPLC-ECD system. The results demonstrated that neurons were significantly damaged by hypoxia for 24 h. When pretreated with gastrodin (100, 200 microg/mL) in hypoxia, neuron survival was significantly increased compared with no gastrodin treatment. Moreover, the enhancement of extracellular glutamate level stimulated by hypoxia was inhibited by pretreatment with gastrodin (100 microg/mL). Further studies demonstrated that gastrodin prevented glutamate- and NMDA-induced neurotoxicity. In addition, gastrodin also inhibited the extracellular glutamate level induced by NMDA insult. These findings suggest that gastrodin has a neuroprotective action against hypoxia in the cultured cortical neuron, and the mechanism may involve a decreasing of the extracellular glutamate level.