Objective To prepare an interfering adenovirus targeting zinc finger and BTB domain containing 20 (ZBTB20) gene. Methods The human and mouse interference primers were designed based on the ZBTB20 gene sequence, and cloned into BamH Ⅰ and Hind Ⅲ restriction endonuclease sites of the adenovirus shuttle vector pShuttle-U6-GFP. The shuttle plasmid pShuttle-shZBTB20 was linearized by Pme Ⅰ and then transformed into pAdEasy-1/BJ5183 bacterial cells for generating homologous recombinant adenovirus plasmid pAd-shZBTB20. The recombinant adenovirus plasmid was linearized with Pac Ⅰ and transfected into HEK-293 packaging cells. The titer of virus was measured by tissue culture infectious dose 50 (TCID₅₀) assay. The interference efficiency of endogenous ZBTB20 gene was detected in Huh7 cells and liver tissue in the mice. Results The adenovirus vector targeting human and mouse ZBTB20 gene was successfully constructed, and the highly active ZBTB20 interfering adenovirus Ad-shZBTB20 was obtained, with the titer of 3.2×10¹⁰ IU/mL. The expression of endogenous ZBTB20 mRNA was significantly reduced to 20% of the control in human hepatoma cell line Huh7 infected with the interfering adenovirus for 96 h. After 7 d of tail vein injection (1×10¹⁰ VG/mouse), ZBTB20 protein was dramatically down-regulated in the liver, and its downstream target gene α-fetoprotein mRNA level was increased by 203 times. Conclusion The prepared ZBTB20-interfering adenovirus can effectively inhibit the expression of human and mouse ZBTB20 and its biological effects.