Ketamine, though widely used in pediatric anesthesia, may induce cortical neurotoxicity in young patients. This study focused on an in vitro model of rat brain embryonic stem cell (ESC)-derived neurons to investigate the effects of microRNA-107 (miR-107) on ketamine-induced neural injury. Rat brain ESCs were proliferated in vitro and differentiated toward neuronal fate. Ketamine induced neural injury in ESC-derived neurons was inspected by TUNEL and neurite growth assays. Ketamine-induce aberrant miR-107 expression was examined by qRT-PCR. MiR-107 was downregulated in ESCs through lentiviral transduction. Its effect on ketamine-induced neural injury in ESC-derived neurons was then examined. Potential downstream target of miR-107, brain derived neurotrophin factor (BDNF), was inspected by dual-luciferase reporter assay and qRT-PCR. BDNF was knocked down, through siRNA transfection, in NSCs to investigate its functional involvement in miR-107 mediated neural protection in ketamine-injured NSC-derived neurons. Ketamine induced apoptosis, neurite degeneration, and upregulated miR-107 in NSC-derived neurons. Lentivirus-mediated miR-107 downregulation attenuated ketamine-induced neural injury. BDNF was proven to be directly and inversely regulated by miR-107 in NSC-derived neurons. SiRNA-mediated BDNF inhibition reversed the protective effect of miR-107 downregulation on ketamine injury in NSC-derived neurons. MiR-107 / BDNF was demonstrated to be an important epigenetic signaling pathway in regulating ketamine-induced neural injury in cortical neurons.