Polo-like kinase 1 (PLK1) is a key regulator in mitosis and a highly potent target for anti-cancer therapies. Several PLK1 inhibitors have been developed and evaluated for cancer treatment. However, none of them has yet been approved for clinical usage, mostly due to their low response rates in cancer patients. A recent phase I trial reported a 44% partial response rate of onvansertib, a PLK1 inhibitor, in the treatment of patients with -mutated metastatic colorectal cancer, indicating that PLK1 inhibitor might be suitable for the treatment of this specific subtype of cancer. This review summarizes the results of preclinical experiments and clinical trials of PLK1 inhibitors, with colorectal cancer as a focus, in hope of facilitating future investigations in this research field.