The events of the cell cycle, the stages at which the cell proliferates and divides, are facilitated and controlled by multiple signaling pathways. Among the many regulatory enzymes that contribute to these processes is the polo-like kinase (Plk). Plks have been reported to mediate multiple mitotic processes, including bipolar spindle formation, activation of Cdc25C, actin ring formation, centrosome maturation, and activation of the anaphase-promoting complex. To investigate its functions in mammalian cells further, we used the recently developed small interfering RNA technique specifically to deplete Plk1 in cultured cells. We find that Plk1 depletion results in elevated Cdc2 protein kinase activity and thus attenuates cell-cycle progression. About 45% of cells treated with Plk1 small interfering RNA show the formation of a dumbbell-like DNA organization, suggesting that sister chromatids are not completely separated. About 15% of these cells do complete anaphase but do not complete cytokinesis. Finally, Plk1 depletion significantly reduces centrosome amplification in hydroxyurea-treated U2OS cells. These data provide direct evidence that Plk is required for multiple mitotic processes in mammalian cells and their significance is discussed.