Polo-like kinases (Plks) play pivotal roles in the regulation of cell cycle progression. Plk1, the best characterized family member among mammalian Plks, strongly promotes the progression of cells through mitosis. Furthermore, Plk1 is found to be overexpressed in a variety of human tumors and its expression correlates with cellular proliferation and prognosis of tumor patients. Although all Plks share two conserved elements, the N-terminal Ser/Thr kinase domain and a highly homologues C-terminal region termed the polo-box motif, their functions diverge considerably. While Plk1 is inhibited by different checkpoint pathways, Plk2 and Plk3 are activated by the spindle checkpoint or the DNA damage checkpoint. Thus, Plk2 and Plk3 seem to inhibit oncogenic transformation. Deregulation of Plk1 activity contributes to genetic instability, which in turn leads to oncogenic transformation. In contrast, Plk2 and Plk3 are involved in checkpoint-mediated cell cycle arrest to ensure genetic stability, thereby inhibiting the accumulation of genetic defects. In this review, we shall discuss the roles of Plks in oncogenesis and Plk1 as a target for therapeutic intervention against cancer.