Urokinase plasminogen activator (uPA) correlates closely with breast cancer metastasis via triggering the degradation of divergent matrix proteins. Here, uPA was selectively knocked down in breast carcinoma MDA-MB-231 cells by siRNAs. The in vitro migration of MDA-MB-231 cells was effectively suppressed accompanied by a decrease in extracellular MMP-9 activities. The colony formation ability of MDA-MB-231 cells was inhibited following uPA knockdown, while the proliferation was not affected. The uPA knockdown in MDA-MB-231 cells caused significantly suppressed tumor metastasis in nude mice. Thus, siRNAs targeted to uPA have implications in the development of novel approaches to preventing breast cancer metastasis.