Colorectal cancer (CRC) is the second most lethal cancer type, with a high incidence rate among adults. The CRC is a highly heterogenic disease with a high rate of mutations in different molecular pathways. Moreover, resistance to standard treatment options is seen frequently among CRC patients. Therefore, a better understanding of the mechanisms behind the initiation, progression, and drug resistance allows us to increase the life quality of CRC patients. TBK1 is a kinase protein with central roles in most cellular signaling pathways. This protein has been reported to be an oncogene in some cancer types. However, the role of TBK1 in colorectal cancer is not yet established. In this study, we generated stable TBK1 knockdown CRC cell lines and mainly focused on the role of TBK1 in colorectal cancer pathogenesis. Upon TBK1 depletion in CRC, we observed increased cell proliferation and migration in vitro. The role of TBK1 in cell proliferation is further confirmed in xenograft models in vivo. Moreover, a shift in EMT and resistance to Gefitinib, an EGFR inhibitor, is indicated in TBK1 knockdown cells. TBK1 is also diminished in our Gefitinib-resistant CRC cell lines, suggesting a role for this protein in drug resistance. The findings of this study suggest a tumor-suppressive role for TBK1 in CRC. Hence, further investigations on the mechanisms behind this tumor-suppressive role of TBK1 in CRC will pave the way for developing novel therapeutic options for CRC treatment.