Objective To construct brain derived neurotrophic factor (BDNF) recombinant plasmid and transfect neural stem cells (NSCs), and observe its effect on differentiation of NSCs. Methods Rat cortical neural stem cells were cultured and identified for transfection. Recombinant plasmid with BDNF gene was constructed and identified by enzyme digestion and sequencing analysis. Non-liposome transfection pattern was used to transfect NSCs. Cells were divided into pcDNA3-1-BDNF group, pcDNA3-1 group and NSCs group without transfection. The expression of BDNF protein and mRNA were detected in infected NSCs by immunocytochemistry and RT-PCR. The infected NSCs were differentiated in medium containing 5% fetal calf serum, and identified by immunoreactive to special antibody, βIII-tubulin, glial fibrillary acidic protein (GFAP) and synaptophysin (SYP). The expression of SYP was observed during the differentiation of NSCs transfected with pcDNA3-1-BDNF. Results NSCs obtained in vitro were nestin positive. Recombinant plasmid with BDNF gene was successfully constructed. The expression of BDNF protein and mRNA of NSCs was significantly increased in pcDNA3-1BDNF group compared to the pcDNA3-1 group and NSCs group was found (EM>P/EM>0.05), but no significant difference between pcDNA3-1 group and NSCs group(EM>P/EM>＞0.05). The cells differentiated into βIII-tubulin positive and GFAP positive cells in all groups, to a lesser extent, also SYP positive in pcDNA3-1BDNF group at 4 days after differentiation. The proportion of βIIItubulin positive for neurons was significantly higher in pcDNA3-1-BDNF group than that in the pcDNA3-1 group and NSCs group at 7 days after differentiation (EM>P/EM>0.05), the number of SYP positive cells and the fluorescence intensity were increased in pcDNA3-1-BDNF group. Conclusion NSCs transfected with recombinant plasmid pcDNA3-1-BDNF have the capacity of producing BDNF, to promote direct