Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer-related death worldwide. The treatment of metastatic CRC considered palliative for many years aiming for an improved life, with little hope of a cure, highlighting the need for developing novel targeted therapy for CRC. Human protein kinases constitute a complicated system with complex internal and external interactions, which stimulates various cellular processes such as cell growth, metabolism, survival, and apoptosis. This study investigated the effect of a combination of atypical Protein Kinase C (PKC) inhibitor (either ICA-I or ζ-Stat) and 5-FU (a thymidylate synthase inhibitor) on CRC cells viability concerning cellular DNA damage. In this study, we took multiple approaches such as colony formation assay, flow cytometry, DNA ladder assay, TUNEL assay, etc. to examine the CRC cell viability and apoptosis as a function of combination treatment. Our findings showed that the combination of atypical PKC inhibitor and 5-FU synergistically reduced the viability of CRC cells and induced apoptosis. Additionally, the DNA ladder and TUNEL assays indicated that there was a notable DNA damage and fragmentation because of lack of thymidylate synthase and due to the deactivation of atypical PKC dependent CDK7. These data suggest that the simultaneous knockdown of upstream atypical PKC protein and downstream DNA damage repairing mechanism would be a useful approach to combat CRC and to improve overall patients’ survival rate.