Polo-like kinase (Plk)1 executes several essential functions to promote cell division. These functions range from centrosome maturation in late G2 phase to the regulation of cytokinesis, which necessitates precise separation of Plk1-dependent substrate phosphorylation over time. Multiple levels of control are in place to ensure that Plk1-dependent phosphorylation of its various substrates is properly coordinated in time and space. Here, we review the current knowledge on the mechanisms that enforce the temporal and spatial control of Plk1 activity, and how this results in coordinated phosphorylation of its many different substrates. We also review a number of newly discovered functions of Plk1 that provide more insights into the spatiotemporal control of Plk1-dependent substrate phosphorylation.