Objective:To construct short hairpin RNA(shRNA) eukaryotic expression vectors selectively silencing β-catenin gene. Methods:Three oligonucleotides targeting of β-catenin gene were synthesized and cloned into eukaryotic expression plasmids pGPU6. Three combinant plasmids pGPU6-β-catenin-shRNA -1,2,3 were transfected into AGS cells by lipofectamine reagent. The gene silencing efficiency was measured by Western blotting. Results:Three shRNA expression vectors were confirmed by restricted endonuclease analysis and partial nucleotide sequencing. pGPU6-β-catenin-shRNA-3 knocked down the expression of β-catenin protein dramatically by Western blot. Conclusion:The eukaryotic vectors expressing shRNA of β-catenin were constructed successfully. The results of this study lay the foundation for further studying on the role of Wnt/β-catenin signaling pathway in gastric cancer.