The protective effect of zoanthamine on Alzheimer’s disease by enhancing differentiation of neural stem cells (NSCs) was evaluated. NSCs were isolated from C57BL/6 mice and assessed by cell viability and apoptosis assays. The cells were separated into five different groups: green fluorescent protein (GFP; transfected with GFP), amyloid precursor protein (APP; transfected with APP), APP + zoanthamine, APP + miR-9 inhibitor, and APP + miR-9 inhibitor + zoanthamine. The effects of zoanthamine on the differentiation of NSCs was determined. Moreover, the effects of zoanthamine on the expression of miR-9 and Notch signalling pathway members was assessed by western blot analysis and reverse-transcription polymerase chain reaction. There was a significant increase in cell viability and a decrease in apoptosis of NSCs in the APP + zoanthamine group compared with the APP group. Treatment with zoanthamine attenuated miR-9 expression and neuronal cell differentiation in APP-treated NSCs. Moreover, in the APP + miR-9 inhibitor group, neuronal cell differentiation and miR-9 expression were significantly reduced, and treatment with zoanthamine reduced the number of differentiated cells and miR-9 expression compared with the APP + miR-9 inhibitor group. There was a significant reduction in the expression of Hes1 and NICD proteins in the APP + zoanthamine group relative to the APP group. In addition, the levels of Hes1 and NICD were enhanced by inhibition of miR-9 but zoanthamine prevented these increases. In conclusion, these results suggest that treatment with zoanthamine enhances the differentiation of NSCs by regulating Notch signalling via elevated miR-9 expression. © 2019, E-Century Publishing Corporation. All rights reserved.