【Abstract】 Objective To construct an adenoviral vector targeting hypoxic inducible factor-1α (HIF-1α ) gene in mouse and identify the effect of it on the expression of HIF-1α in cultured mouse embryonic neural stem cells, and provide the basis for investigating the contribution of HIF-1α signaling pathway in neural stem cell biology. Methods The HIF-1α template DNA sequence was designed by using bioinformatics. DNA recombinant technique was used to construct recombinant adenoviral vector, AdHIF-1α-shRNA-EGFP. Polymerase chain reaction (PCR) and sequencing were performed. Neural stem cells from frontal cortex of mouse were transfected with AdHIF-1α-shRNA-EGFP in vitro. The efficiency of transfection was detected by flow cytometry. The transfected cells were cultured under hypoxia. Double immunofluorescent staining of nestin and HIF-1α was performed and the expression of HIF-1α was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Results The sequence cloned into adenoviral vector was identical with that synthesized chemically. Neural stem cells were transfected effectively by AdHIF-1α-shRNA-EGFP. The results from RT-PCR and Western blot showed that the expression of HIF-1α gene and protein in neural stem cells transfected with AdHIF-1α-shRNA-EGFP decreased by 52.05% and 67.27% under hypoxia, respectively. Conclusion AdHIF-1α-shRNA-EGFP inhibited effectively hypoxia-induced expression of HIF-1α gene and protein in mouse neural stem cells in vitro.