Objective To explore the changes of microRNA expression during the neuronal differentiation of neural stem cells (NSCs) induced by Plastrum testudinis extract (PTE) . Methods NSCs were isolated from the hippocampus of 14-day SD rat fetus and were obtained after primary culture. The abilities of NSCs differenting into neurons were identified by immunofluorescent staining. NSCs were randomly divided into three groups, blank control group, and low- and high-dose PTE groups ( 3, 30 μg/mL PTE) . After the cells were incubated with PTE for 7 days, total RNA was isolated and then the expression of miR-124 and miR-9 was observed by real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR) . Results Compared with the blank control group, the expression of miR-124 and miR-9 remained unchanged in low-dose PTE group, but was significantly increased in high-dose PTE group ( P<0.05). Conclusion PTE promotes NSCs differentiating into neurons, which might be associated with the up-regulation of miR-124 and miR-9.