Objective To construct eukaryotic expression vector of brain-derived neurotrophic factor (BDNF) and detect its effect of overexpression on differentiation of rat neural stem cells (NSCs) into neurons. Methods The RT-PCR was used to amplify rat BDNF gene from RNA of rat hippocampus. The BDNF gene was inserted into eukaryotic expression vector pEGFP-N1 to construct recombinant expression vector pEGFP-N1-BDNF. The recombinant vector was transfected into NSCs by Lipofectamine 2000.The expression of BDNF mRNA in NSCs was detected by RT-PCR. The differentiation of rat NSCs into neurons was detected by immunohistochemistry staining. Results The sequence of the cloned BDNF was confirmed to be correct by DNA sequencing. The NSCs transfected with pEGFP-N1-BDNF expressed BDNF efficiently. The pEGFP-N1-BDNF transfected NSCs differentiated into more neurons than the pEGFP-N1 transfected ones (EM>P/EM>0.01). Conclusion All these results indicate that BDNF overexpression significantly promotes the differentiation of rat NSCs