Ketamine, a commonly used anesthetic compound, may cause neurotoxicity in immature or developing brains. In this study, we indented to understand the molecular expression and functional role of Lin-28 Homolog B (lin28b) in regulating ketamine-induced neurotoxicity in neural stem cells (NSCs)-differentiated neurons. NSCs from embryonic rat brains were cultured in vitro, and induced toward neuronal differentiation. NSCs-differentiated neurons were treated with various concentrations of ketamine for 24 h to evaluate the concentration-dependent effect of ketamine on endogenous lin28b mRNA level. QRT-PCR showed that lin28b was downregulated by ketamine in NSCs-differentiated neurons, in concentration-dependent manner. Neurons were then transfected with adenovirus to ectopically upregulate lin28b. We found that ketamine-induced apoptosis and neurite retraction in NSCs-differentiated neurons were significantly reduced by adenovirus-mediated lin28b upregulation. Expression of sex determining region Y box 2 (Sox2) mRNA was examined in ketamine-injured and lin28b-upregulated NSCs-differentiated neurons. It was found Sox2 was downregulated by ketamine, and overexpressed by lin28b upregulation. Finally, Sox2 was downregulated by siRNA in NSCs-differentiated neurons. And we discovered that Lin28b-upregulation-associated neural protection was severely hampered by Sox2 downregulation in ketamine-injured neurons. Thus, Lin28b and Sox2 are important molecular components in ketamine-induced neurotoxicity.