Modulation of stem cell proliferation is a crucial aspect of neural developmental biology and regenerative medicine. To investigate the effect of optical stimulation on neural stem cell proliferation, cells transduced with channelrhodopsin-2 (ChR2) were used to analyze changes in cell proliferation and cell cycle distribution after light stimulation. Blue light significantly inhibited cell proliferation and affected the cell cycle, which increased the percentage of cells in G1 phase and reduced the percentage in S phase. It is likely that the influence of blue light on cell proliferation and the cell cycle was mediated by membrane depolarization, which induced accumulation of p21 and p27 proteins. Our data provide additional specific evidence that membrane depolarization may inhibit neural stem cell proliferation.