Abstract： Objective To determine the effect of Tenuigenin (TEN) on the Mashl expression in neural stem cells (NSCs) from mouse hippocampus and directional differentiation of its NSCs in vitro. Methods NSCs were generated from the mouse hippocampus within 24 hours after birth and cultured with EGF and bFGF (20 ng/mL, each) in a 50-mm uncoated culture flask. The third-passage NSCs were cultured with TEN medium and divided them into vehicle, 5 mg/L, 20 mg/L and 100 mg/L groups according to different TEN concentrations. Immunocytochemical techniques were employed to detect the NSE-positive neurons and ChAT-positive neurons induced by TEN, and the expression of Mash1 in NSCs affected by TEN was measured by RT-PCR techniques. Results The immunocytochemical and RT-PCR results revealed that ChAT-positive neurons and NSE-positive neurons were detected with statistical significance and 20 mg/L group showed a stronger effect than other concentration groups (P< 0.05). The expression of Mash1 in the 20 mg/L group increased as compared with that in the vehicle group (P<0.05). Conclusion TEN can induce NSCs in vitro differentiating into neurons and cholinergic neurons, and increase the expression of Mashl in NSCs.