Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-β (TGF-β) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-β supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-β at higher concentrations than autocrine TGF-β further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-β is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-β-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-β maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-β further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.