Objective To understand and compare the proportion of neural stem cells (NSCs) in the whole brain and cerebral cortex of mice at different embryonic days, and provide quantitative data for the later optimization of NSCs isolation and culture. Methods The whole brains (at embryonic 12.5, 14, 16 and 18 days) and cerebral cortex (at embryonic14, 16 and 18 days) were isolated and digested into single cell suspension, and were adherently cultured for 3-4 h.Immunofluorescence staining of Nestin, a NSCs specific marker, was used to statistically analyze the proportion of NSCs in each group.Expression of Nestin mRNA in the cerebral cortex of mice at E12.5, E14, E16, and E18 was detected by real-time fluorescence quantitative PCR. Results The result of immunofluorescence assay showed that there were Nestin-positive cells in the whole brain and cerebral cortex of mice at different embryonic days. In the whole brain,the proportion of NSCs was highest at E12.5 (53.42±1.57%) and lowest at E18(25.96±1.31%), and the proportions at E14 and E16 were placed in the middle among the groups. In the cerebral cortex, the highest proportion of NSCs was at E14 (33.65±0.29%), and the lowest at E18(25.29±0.28%), and the middle at E16 (26.82±0.30%).The result of real-time PCR showed that when the mRNA expression of Nestin in the cerebral cortex was set to 1, the relative mRNA expression of Nestin was 0.83±0.04 at E14, 0.77±0.05 at E16, and 0.44 ±0.05 at E18. Thus, the mRNA expression level of Nestin in the mouse cerebral cortex was gradually decreasing with the increase of embryonic days. Conclusions During the brain development, the proportion of NSCs is gradually decreasing in the whole brain and cerebral cortex of mice with the increase of embryonic days.