Although stem cells can proliferate and differentiate through the completion of cell cycle progression, little is known about the genes and molecular mechanisms controlling this process. Here, we investigated the effect of the inhibition of cell cycle by cyclin D1 gene knockout on proliferation and differentiation of neural stem cells (NSCs). Knockout of cyclin D1 induced the cultured neural stem cells arrested at the G0/G1 phase as detected by flow cytometry. Cyclin D1 knockout led to the apoptosis of NSCs and inhibited the differentiation into astrocytes without affecting the differentiation into neurons. We further demonstrated that a significant reduction of BrdU(+) cells in the subgranular zone of the dentate gyrus and subventricular zone was found in cyclin D1 gene knockout (cyclin D1(-/-)) mice compared with cyclin D1(+/+) and cyclin D1(+/-) mice. These observations demonstrated that cyclin D1 plays essential roles in the proliferation and differentiation of neural stem cells.