The invention discloses a ZNF667 protein short hairpin RNA (ribonucleic acid) interference plasmid and an application method thereof. The ZNF667 protein short hairpin RNA (ZNF667-shRNA) interference plasmid is successfully constructed at the first time and transfected into a hepatoma cell line HepG2, and a cell line for stably transfecting the ZNF667-shRNAinterference plasmid is constructed. Proliferation, infiltration, invasion and migration of HepG2 cells can be inhibited by reducing ZNF667 protein in the HepG2 cells. Nude mouse transplantation tumor experiments confirm that tumor formation can be inhibited from an in-vivo level by reducing ZNF667 expression in the HepG2 cells. The malignant degree of the HepG2 cells is inhibited possibly by promoting P53 and inhibiting Bcl-2 protein in reduction of ZNF667 protein expression in the HepG2 cells. A new clue and path can be provided for treating liver cancer, and the interference plasmid is of great significance.