BACKGROUND/AIMS: Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. METHODS: Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. RESULTS: Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. CONCLUSION: PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.