Objective Investigation of the effect of dexmedetomidine on the changes induced by midazolam in proliferation, differentiation and apoptosis of neural stem cells (NSCs) from rats in vitro. Methods NSCs were isolated from the cerebral cortex of rat embryos on embryonic day 14-15, and P1-P2 cells were treated with midazolam (10μM), dexmedetomidine (1μM) combined with midazolam (10μM) 24 h , with the untreated cells served as control. Cell viability was detected by MTT assay. Cell proliferation was evaluated by 5’-bromo-2’-deoxyuridine (BrdU) incorporation. Immunocytochemical staining was used to study the differentiation of NSCs. Cell apoptosis was assessed by terminal dUTP nick-end labeling (TUNEL). Results Treatment with midazolam significantly inhibited cell viability (Control group 0.214±0.006, Mid group 0.187±0.002，P<0.01) and proliferation (Control group 35.7%±1.0%, Mid group 27.6%±1.0%, P<0.01), increased the cell apoptosis (Control group 5.7%±0.8%，Mid group 7.8%±1.1%, P<0.01), but did not affect the differentiation of NSCs (P>0.05). Compared with midazolam group, the combination of dexmedetomidine and midazolam significantly improved cell viability (0.233±0.007，P<0.01) and proliferation (35.7%±1.1%), and decreased the cell apoptosis (5.3%± 1.0%, P<0.01). There was no significant difference in the differentiation of NSCs between the midazolam group and the combination group (P>0.05). Conclusions Treatment with dexmedetomidine improves the decreased NSCs proliferation and increased cell apoptosis induced by midazolam, but has no significant influence on differentiation of NSCs into neurons and astrocytes.