Colorectal malignancies associated with KRAS and TP53 mutations led us to investigate the effects of combination therapy targeting KRAS, MEK1, or PLK1 in colorectal cancer. MEK1 is downstream of RAS in the MAPK pathway, whereas PLK1 is a mitotic kinase of the cell cycle activated by MAPK and regulated by p53. Bioinformatics analysis revealed that patients with colorectal cancer had a high expression of MAP2K1 and PLK1. Furthermore, PLK1 and MEK1 activity in human colorectal adenocarcinoma (COAD) tissues was found to be highly upregulated compared to healthy tissues. To determine the sensitivity of KRAS or/and TP53-mutated cancer to KRAS, MEK1, or PLK1-targeted therapy, the inhibitors salirasib, trametinib, volasertib, and onvansertib were used in COAD cells with different KRAS and TP53 status. The results showed that combinations with trametinib and PLK1 inhibitors were more potent than combinations with salirasib. A combination of MEK1 and PLK1 inhibitors exhibited significant therapeutic effects on KRAS or/and TP53-mutated COAD cells. Notably, the combination of trametinib and onvansertib effectively suppressed tumor growth in a xenograft mouse model of KRAS and TP53-mutated COAD. This treatment induced G1 and G2/M arrest, respectively, and showed the strongest synergistic effect in KRAS and TP53-mutated SW48 cells expressing mutant KRAS^G13D and transduced with TP53 shRNA, ultimately leading to apoptotic cell death. These effects are attributed to two-step inhibition mechanism that blocks the MAPK signaling pathway and disrupts mitosis in KRAS and TP53-mutated COAD cells.