Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate.