Polo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. New techniques, including the application of small-molecule inhibitors, have greatly expanded our knowledge of the functions, targets, and regulation of this key mitotic enzyme. In this review, we focus on how Plk1 is recruited to centrosomes, kinetochores, and the spindle midzone and what the specific tasks of Plk1 at these distinct subcellular structures might be. In particular, we highlight new work on the role of Plk1 in cytokinesis in human cells. Finally, we describe how better understanding of Plk1 functions allows critical evaluation of Plk1 as a potential drug target for cancer therapy.