Objective To investigate the effect of berberine on proliferation of neural stem cells (NSCs) induced by hydrogen peroxide (H₂ O₂ ). Methods NSCs from Sprague-Dawley rats were isolated and purified by suspension culture. Cells were divided into a control group, H₂ O₂ group (NSCs exposed to H2 O2 injury), berberine group (NSCs were incubated with berberine concentrations ranging from 0.5 to 20 μmol/ L and exposed to H₂ O₂ ), and DAPT (a blocker of the Notch signaling pathway) group. Cell viability was evaluated using the Cell Counting Kit-8 assay. Proliferation of NSCs was evaluated by a neurosphere formation assay and Ki67 protein expression. Expression of key proteins in the Notch signaling pathway (including notch1 and hes1) in response to berberine treatment or DAPT (a Notch inhibitor) was determined by Western blotting. Results Cell viability of NSCs was significantly increased by berberine compared with the H₂ O₂ group. The neurosphere growth assay showed that 5 or 10 μmol/ L berberine increased NSC proliferation. The ratio of Ki67 ⁺ / DAPI cells and notch1 and hes1 protein expression increased significantly compared with the H₂ O₂ group. Conclusions Berberine treatment upregulates Notch signaling in NSCs, whereas DAPT attenuates these effects. Berberine is a drug that promotes NSC proliferation and exerts a protective effect on NSCs via the Notch signaling pathway.