It has been reported that a prior exposure of isoflurane, a commonly used volatile anesthetic in clinical practice, reduces brain cell death after ischemia. This isoflurane preconditioning-induced neuroprotection has been shown in rat in vivo and in vitro brain ischemia models. To investigate the mechanisms of this protection, we used the human neuroblastoma SH-SY5Y cells and simulated ischemia in vitro by oxygen-glucose deprivation. We found that isoflurane exposure for 30 min at 24 h before a 5-h oxygen-glucose deprivation dose-dependently reduced cell death. Isoflurane exposure induced phosphorylation/activation of extracellular signal-regulated kinase (ERK). Inhibition of the phospho-ERK expression abolished the isoflurane preconditioning-induced protection. Isoflurane exposure also increased the expression of early growth response gene 1 (Egr-1) and Bcl-2, proteins downstream of ERK. Egr-1 is a transcription factor and plays a role in cell survival. Bcl-2 is an anti-apoptotic protein. The increased expression of Egr-1 and Bcl-2 by isoflurane was inhibited by ERK inhibition. Thus, our results suggest a role of ERK/Egr-1/Bcl-2 pathway in the isoflurane preconditioning-induced protection in the human neuroblastoma SH-SY5Y cells.