Abstract: Objective To observe the effects of Naoluo Xintong on the expression of β-tubulin III and glial fibrillary acidic protein (GFAP) and the proliferation and differentiation of murine neural stem cells (NSCs) in vitro. Methods An immortalized murine NSC line was divided into model control (MC) group, 10% Naoluo Xintong drug-containing serum group (NLXT group), and 10% Naoluoxintong drug-containing serum with inhibitor Y27632 group (Y-27632 group) with corresponding treatments. The activity of the NSCs was detected after the treatments using MTT assay, and the migration of the cells was observed with Transwell assay. The expressions of β-tubulin III, GFAP and MAP-2 proteins in the cells were detected with immunoblotting, and the expressions of DCX, NEUN, and β-tubulin III were also detected with immunofluorescence assay. Results Compared with that in MC group, the number of migrated cells in NLXT group and Y-27632 group increased significantly at 1 day and 3 days after induction (P<0.05). The survival rate and the number of migrated cells in NLXT group and Y-27632 group increased significantly on day 7 (P<0.01). Compared with those in MC group, the expressions of β-tubulin III, MAP2 and GFAP protein in NLXT group and Y-27632 group were significantly increased on days 3 (P<0.01) and 7 (P<0.05). The numbers of β-tubulinIII/ GFAP, BrdU/DCX, and BrdU/NEUN labeled cells in the NLXT group and Y-27632 group were significantly greater than those in the MC group. Conclusion Naoluo Xintong promotes the proliferation and differentiation of murine NSCs in vitro by regulating the expressions of β-tubulinIII/GFAP.