Abstract: Though melatonin has a wide variety of biological functions, its effects on the neural stem cells (NSCs) is still unknown. In this study, we examined the effects of melatonin at either physiological (0.01–10 nm) or pharmacological concentrations (1–100 μm) on the proliferation and neural and astroglial differentiation of NSCs derived from the mouse embryo striatum using an in vitro culture system. We found that melatonin at pharmacological concentrations, but not at physiological concentrations, suppressed epidermal growth factor (EGF)-stimulated NSC proliferation (increment of viable cells, DNA synthesis and neurosphere formation) in a concentration-dependent manner. Furthermore, treatment with melatonin at a pharmacological concentration during the proliferation period facilitated 1% FBS-induced neural differentiation of NSCs without affecting the astroglial differentiation. In contrast, the treatment with melatonin at pharmacological concentrations during the differentiation period decreased the neural differentiation of the NSCs. As with melatonin, MCI-186, an antioxidant, suppressed EGF-stimulated NSC proliferation and facilitated the subsequent neural differentiation of NSCs. These results suggest that melatonin exerts potent modulatory effects on NSC functions including the suppression of the proliferation and facilitation of neuronal differentiation, likely via its antioxidant activity. As neurogenesis is thought to play an important role in ameliorating the deficit in neurodegenerative diseases, melatonin might be beneficially used for the treatment diseases such as cerebral infarction.