Prostate cancer (PCa) is one of the most common cancers in men. Each year approximately 543,000 new cases are reported worldwide, and the disease kills 200,000 (mostly older men) in developed countries. The existing treatment approaches and surgical intervention have not been able to effectively manage this dreaded cancer and, therefore, continuing efforts are ongoing to explore novel targets and strategies for the management of PCa. The activity of polo-like kinase 1 (Plk1) is elevated in tissues and cells with a high mitotic index, including cancer cells. An increasing body of evidence suggests that the level of Plk1 expression has prognostic value for predicting outcomes in patients with some cancers. A close correlation between Plk1 expression and carcinogenesis has been documented. However, the role of Plk1 in PCa is not known. We propagated a hypothesis that Plk1 inhibition will result in elimination of human PCa cells via a mitotic arrest followed by apoptosis (1). To define the role of Plk1 in PCa, we used the technique of RNA silencing via small interfering RNA (siRNA). First, using a series of human prostate carcinoma cells and normal human prostate epithelial (PrEC) cells, we assessed Plk1 levels in PCa. Immunoblot analyses clearly showed a significant expression of Plk1 in LNCaP, DU145, and PC3 human PCa cells. Interestingly, Plk1 was not detectable in normal PrEC cells. Next, we transfected the PCa cells with Plk 1 siRNA, which resulted in a significant inhibition in Plk1 protein in all PCa cells. Plk1 depletion resulted in a decrease in cell viability and induction of apoptosis in PCa cells but had no appreciable effect in normal PrEC cells. Our data also demonstrated that Plk1 siRNA transfection of PCa cells resulted in 1) a mitotic cell cycle arrest, 2) failure of cytokinesis, and 3) defects in centrosome integrity and maturation. Thus, our study suggested that 1) Plk1 plays a critical role in the process of PCa development and 2) gene therapeutic approaches aimed at Plk1 or the pharmacological inhibitors of Plk1 may be developed for the management of PCa.