MDCK is the main cell line for influenza vaccine production. Previous studies have reported that MDCK cells have tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, more caution is needed in vaccine production for human use. Therefore, the use of gene editing technology to establish cells that cannot form tumors can significantly improve the biosafety of influenza vaccines. The key is to understand the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells. However, our understanding is still superficial. We previously obtained a cell line CL23 with significantly reduced cell proliferation, migration, and invasion through a monoclonal cell screen, and subsequent tumor-bearing experiments in nude mice showed no tumorigenic cells. DIA proteomics method was used to compare the protein expression differences between wild-type (M60) and non-tumorigenic (CL23) cells, and to explore the genes related to tumorigenesis in MDCK cells. The differentially expressed proteins were verified at the mRNA level by RT-qPCR, and several genes involved in cell tumorigenesis were preliminarily screened. Western blot further confirmed that the expression of related proteins was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of influenza virus. Therefore, CDC20 is an effective target gene for inhibiting the tumorigenicity of MDCK cells. This study lays the foundation for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.