Preconditioning neurons with isoflurane, a commonly used volatile anesthetic in clinical practice, improves tolerance of subsequent ischemia in both intact animal models and in vitro preparations. To investigate the mechanisms of this protection, we primarily cultured rat hippocampal neurons and simulated ischemia in vitro by oxygen-glucose deprivation (OGD). Neuron viability was measured. Neuron injury was observed by inverted phase contrast microscope and assessed by lactate dehydrogenase (LDH) release. Gene expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Isoflurane exposure for 2 h at 24 h before a 2 h OGD dose-dependently reduced cell injury. Isoflurane accumulated phosphorylation/activation of extracellular signal-related kinases 1 and 2 (Erk1/2) and hypoxia inducible factor (HIF)-1alpha, a transcription factor involved in cell survival. Inhibition of the phospho-Erk1/2 partially abolished the isoflurane preconditioning-induced HIF-1alpha protein content accumulation and neuroprotection. Isoflurane also increased inducible nitric oxide synthase (iNOS) mRNA levels, a downstream gene of HIF-1alpha. Thus, the current results indicate that isoflurane preconditioning activates HIF-1alpha during protection against OGD neuronal injury and the activation might be partly mediated by the Erk1/2 pathway.