Earlier studies show that Hes1 expression is oscillatory in neural stem cells but sustained and high in the roof plate and the floor plate, and that such different dynamics of Hes1 expression (oscillatory versus sustained) regulate different proliferation and differentiation characteristics of these cells (active in neural stem cells but rather dormant in roof/floor plate cells). The mechanism of how different dynamics of Hes1 expression is controlled remains to be determined. Here, we found that the seed sequence of microRNA-9 (miR-9) is complementary to the 3'-UTR sequence of Hes1 mRNA. MiR-9 is highly expressed in the ventricular zone of the developing brain, which contains neural stem cells, but it is not expressed in the roof plate or the floor plate. Over-expression of miR-9 negatively regulates the Hes1 protein expression by interacting with the 3'-UTR of Hes1 mRNA, thereby inducing cell cycle exit and neuronal differentiation. Conversely, knockdown of miR-9 inhibits neuronal differentiation. Furthermore, knockdown of miR-9 inhibits the oscillatory expression of Hes1 mRNA in neural stem cells. These results indicate that miR-9 regulates the proliferation and differentiation of neural stem cells by controlling the dynamics of Hes1 expression in the developing brain.