Purpose: Notch, a type 1 transmembrane protein, plays a key role in the development of many tissues and organ types. Aberrant Notch signaling, found in a wide variety of human cancers, contributes to tumor development. Because Notchl was found to be overexpressed in prostate cancer (PCa) cells and human PCa tissue, we therefore tested our hypothesis that overexpression of Notchl in PCa promotes tumor invasion. Experimental Design: Notchl expression was evaluated in human PCa cells and human PCa tissues. PCa cells were transiently transfected with Notchl-specific small interfering RNAs in concentrations ranging from 30 to 120 nmol/L and subsequently evaluated for effects on invasion and expression analysis for molecules involved in invasion. Results: Small interfering RNA-mediated knockdown of Notchl in PC3 and 22Rv1 PCa cells dramatically decreased their invasion. Focused cDNA array revealed that Notchl knockdown resulted in significant reduction in the expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) gene transcripts. These data were further verified by reverse transcription-PCR, real-time reverse transcription-PCR, and immunoblot analysis. Knockdown of Notchl was also observed to significantly reduce the mRNA expression and protein levels of uPA and its receptor uPAR. A significant reduction in MMP9 expression in Notchl knockdown cells suggested a role for Notchl in augmenting MMP9 transcription. Conclusions: Our data show the involvement of Notchl in human PCa invasion and that silencing of Notchl inhibits invasion of human PCa cells by inhibiting the expression of MMP9 and uPA. Thus, targeting of Notchl could be an effective therapeutic approach against PCa.