Methylmercury (MeHg), a cause of Minamata disease, has been reported to act as a neurotoxic agent during brain development and to produce fetal Minamata disease, which is characterized by brain malformation and malfunction. We have reported that proliferation of neural stem cells (NSCs), prepared from mouse and monkey embryonic stem (ES) cells by the Neural Stem Sphere (NSS) method, is dose-dependently inhibited by MeHg. In this study, effects of MeHg on differentiation of monkey NSCs were investigated. When the NSCs were induced to differentiate into neurons in the presence of MeHg (from 100 nM to 3 μM), the cell number was dose-dependently decreased. In addition, the cells differentiating into neurons and the neurons differentiated from the NSCs were demonstrated to be more susceptive to MeHg than the proliferating NSCs. The effects of MeHg on the cells were supported by gene expression analysis using RT-PCR and immunofluorescent analysis. These results suggest that MeHg inhibits both proliferation and neuronal differentiation of the NSCs and that it consequently causes neurotoxic effects in the development of central nervous system.