Objective To determine the expression profiles of main molecules in Wnt signal pathway, β-catenin and glycogen synthase kinase-3β (GSK-3β), in the proliferation and differentiation process of neural stem cells (NSCs), and to investigate the possible regulating role of these molecules in the process. Methods The NSCs were isolated and primarily cultured from rat fetus at an embryonic age of 14 to 16d according to reported methods. At 12 to 24h after cultured in the medium containing 10% fetal bovine serum (FBS), the cells attached and grew gradually. Western blotting was used to measure the protein levels of β-catenin and GSK-3β in the cells treated in the FBS medium or control after 12, 24, 48 and 72 hours treatment. Results Immunofluorescence assay indicated that the cultured neurospheres or single cells were positive with nestin. Western blotting results revealed a continuous expression of β-catenin and GSK-3β in the differentiation of the cultured NSCs. β-catenin was in a gradual decreasing tendency, whereas GSK-3β showed an opposite change. Conclusions Wnt signaling pathway is involved in the proliferation and differentiation of NSCs, and is active first and then gradually inhibited. These confirm the NSCs biological property of the culturing cells and provide a model for studying the role of Wnt signaling molecules.