〔Abstract〕 Objective To explore whether miR-9 affects the proliferation of neural stem cells(NSCs) by regulating β-tubulin Ⅲ and glial fibrillary acidic protein(GFAP). Methods NSCs cells were isolated and cultured on the purchased healthy pregnant mice and the isolated cells were divided into NO group(non-transfected NSCs cell line), NN group(NSCs transfected with miR-9-NC), NM group(NSCs transfected) transfected with miR-9 mimics), NI group(transfected with miR-9 inhibitor). Nestin was identified by immunofluorescence, the contents of Mir-9, β-tubulin Ⅲ and GFAP were measured by QRT-PCR, the proliferation of NSCs was measured by MTT method, the apoptosis of NSCs was measured by flow cytometry, and the protein expressions of Mir-9, β-tubulin Ⅲ and GFAP were detected by Western blot. Results The detection index Nestin of NSCs for 24 h after cell passage was positive in 90% of the NSCs, and co-localized with the nucleus, indicating the successful isolation of NSCs. The expression of miR-9 mRNA in NSCs of the NM group was the highest among the four groups, indicating that the transfection was successful. Among the four groups, β-tubulin Ⅲ mRNA and protein expression was the highest in the NM group, and the GFAP mRNA and protein expression was the lowest. The NI group was the opposite(P<0.05). In the four groups after 24 h, the cell activity of NSCs in the NM group was the strongest and showed a trend of gradually increasing with time(P<0.05). The cell activity of NI was significantly weaker than that of the other three groups(P<0.05). Among the four groups, the apoptosis rate of the NM group was lower than that of the other three groups(P<0.05), and the apoptosis rate of NI was higher than that of the other three groups(P<0.05). The difference in the above indicators between the NO group and the NN group was smaller. Conclusion Overexpressed miR-9 promotes the proliferation of NSCs by promoting the expression of β-tubulin Ⅲ and inhibiting the expression of GFAP.