Objective:To investigate whether the effect of fluoxetine on cell proliferation involving Notch1 signaling in cultured neural stem cells (NSCs). Methods:The MTT assay was used to evaluate cell viability. The protein level of Notch1 signaling components (including NICD,Hes1 and Hes5) were detected by Western blot. Results:①When the cells were treated with the 10 μmol/L,15 μmol/L and 20 μmol/L fluoxetine for 48 h,the cell viabilities increased significantly more than those without fluoxetina treated. ②MTT assay showed the presence of fluoxetine strongly enhanced the cell proliferation compared to the control,while DAPT decreased cell proliferation. When fluoxetine and DAPT in combination were added,the cells of proliferation were declined compared with single fluoxetine treated group. ③ Inactivation of Notch signaling with DAPT lead to a rapid reduction in the protein expression level of Hes1and Hes5 compared with the control. After the treatment of fluoxetine for 48 h,the protein expressions of NICD,Hes1 and Hes5 increased significantly compared with the control. When NSCs were exposed to fluoxetine-conditioned medium with DAPT,western blot analysis revealed that the levels of NICD,Hes1 and Hes5 were decreased relative to fluoxetine-conditioned medium without DAPT. Conclusion:①Fluoxetine enhanced cell proliferation in cultured NSCs. ②Fluoxetine activated the Notch1 signaling pathway in NSCs. ③ Notch1 signaling play a major role in the proliferation of NSCs promoted by fluoxetine.