This study was aimed to construct a recombinant adenovirus vector for antisense klf4 gene through AdEasy system. Human klf4 cDNA was reversely inserted into the multiple cloning sites (MCS)of the pShuttle-CMV by using the backbone plasmid AdEasy-l, the antisense klf4 gene was constructed through homologous recombination in E.coli BJ5183, then the adenoviruses were packaged and amplified in the HEK 293 ce1ls. The adenovirus vector for antisense klf4 gene confirmed by PCR, restriction analysis and DNA sequencing. After being transfected with the adenoviruses at 200 MOI for 48 hours, total RNA and protein were extracted from human umbilical vein endothelial cells (HUVEC). Klf4 mRNA and KLF4 protein expression levels were evaluated by Real-time PCR and Western-blot. The results showed that the recombinant adenovirus vector for antisense klf4 gene was successfully constructed, recombinant adenovirus could suppress the expression of klf4 mRNA and KLF4 protein in HUVECs. It is concluded that the adenovirus vector for antisense klf4 gene has been constructed successfully, which provides the material basis for further studying the biologic function and potential application of klf4.