Objective To construct lentivirus vectors with co-expression of mouse Wnt3a(mWnt3a) gene and green fluorescent protein (GFP), transfect into neural stem cells (NSCs), and observe the expression of mWnt3a in NSCs. Methods Gene recombinant technology was employed to clone mWnt3a gene to lentivirus vector pLVX-IRES-ZsGreen1 to construct a lentiviral vector pLVX-Wnt3a-IRES-ZsGreen1. Lentiviral vectors were packaged and the titer was determined. NSCs were transfected with the constructed pLVX-Wnt3a-IRES-ZsGreen1 (Wnt3a-NSCs group) or pLVX-IRES-ZsGreen1 (GFP-NSCs group), with normal NSCs (NSCs group) as controls. NSCs of Wnt3a-NSCs group were identified by immunoflourence staining for nestin, the expression of mWnt3a mRNA in NSCs of each group was detected by Real-Time PCR, and Western blotting was applied to detect the expression of mWnt3a and β-catenin protein in NSCs of each group. Results It was confirmed by restriction enzyme digestion, DNA sequencing analysis and GFP expression detection that recombinant pLVX-Wnt3a-IRES-ZsGreen1 vector was successfully constructed, with virus titer up to 3×10⁸ TU/mL. Not only green fluorescence but also expression of nestin were observed in NSCs of Wnt3a-NSCs group. Real-Time PCR and Western blotting revealed that the expression of mWnt3a mRNA and protein and β-catenin protein in Wnt3a-NSCs group was significantly higher than that in GFP-NSCs group and NSCs group 7 d after transfection (P<0.01). Conclusion The recombinant lentiviral vector carrying mWnt3a can be successfully constructed, and mWnt3a gene can be successfully transfected into NSCs in vitro.