Purpose: Overexpression of transforming growth factor (TGF) -β has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-β1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management. Experimental Design: Small interfering RNA constructs targeting TGF-β1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis. Results: Inhibition of TGF-beta;1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-β signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKTand extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-β receptor expression showed a decrease in both receptor I and II expression in TGF-β1 silenced cells. These results suggest that inhibition of TGF-β1 ligand may act as a negative feedback loop to disrupt the function of all TGF- β isoforms. Conclusions: Therapies targeting the TGF-β signaling pathway may be more effective in latestage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulatingTGF-β levels. © 2008 American Association for Cancer Research.