Polo-like kinase 1 (Plk1) is required for multiple stages of mitosis and is up-regulated in many human malignancies. We depleted Plk1 expression using small interfering RNA (siRNA) and showed defects in bipolar spindle formation and cytokinesis, growth inhibition, and apoptosis induction in human cancer cell lines. To our surprise, depletion of Plk1 in normal human cells did not result in obvious cell cycle defects, and did not induce significant inhibition of cell growth for at least two cell cycles. In addition, Plk1 siRNA inhibited colony formation in soft agar and tumorigenesis in a HT1080 xenograft model in a dose-dependent manner. Analysis with isogenic pairs of cell lines, differing in p53 status, revealed that Plk1 depletion preferentially induced mitotic arrest, aneuploidy, and reduced cell survival in the p53-defective cell lines. No obvious defects were observed in most p53 wild-type cells during the first few cell cycles. In addition, long-term survival studies revealed that p53 facilitates survival upon Plk1 depletion. Therefore, short-term inhibition of Plk1 can kill tumor cells while allowing normal cells to survive. These data validate the episodic inhibition of Plk1 as a very useful approach for cancer treatment.