Propofol is an intravenous anesthetic with neuroprotective effects against cerebral ischemia or hypoxia injury. However, the underlying mechanisms remain obscure. Recent years emerging evidence has demonstrated that metallothionein-3 (MT-3), a growth inhibitory factor that exists mainly in the central nervous system, exhibited neuroprotective effect in vivo. Here, we used a model of hypoxia/re-oxygenation (H/R) injury to examine the hippocampal neuroprotective effect of propofol, and explored the role of MT-3 in this action. H/R resulted in reduced cell viability and increased cell death in hippocampal neuron culture, as indicated by MTT assay and lactate dehydrogenase (LDH) release assay, respectively. Pretreatment of propofol at different concentrations (50, 150, and 250 μmol/L) reversed H/R-induced neurotoxicity and increased MT-3 mRNA and protein expressions. Moreover, propofol failed to exert neuroprotective effect when MT-3 was silenced by the transfection with the specific siRNA, suggesting that MT-3 was the crucial mediator for propofol's neuroprotective effect against H/R. In conclusion, our findings showed that propofol is neuroprotective in H/R model on hippocampal neuron cells and that it may act by up-regulation of MT-3.