Polo-like kinase 1 (PLK1) plays an important role in the initiation, maintenance, and completion of mitosis. Dysfunction of PLK1 may promote cancerous transformation and drive its progression. PLK1 overexpression has been found in a variety of human cancers and was associated with poor prognoses in cancers. Many studies have showed that inhibition of PLK1 could lead to death of cancer cells by interfering with multiple stages of mitosis. Thus, PLK1 is expected to be a potential target for cancer therapy. In this article, we examined PLK1’s structural characteristics, its regulatory roles in cell mitosis, PLK1 expression, and its association with survival prognoses of cancer patients in a wide variety of cancer types, PLK1 interaction networks, and PLK1 inhibitors under investigation. Finally, we discussed the key issues in the development of PLK1-targeted cancer therapy.