OBJECTIVE: Targeted down-regulation of TGF-β expression inhibits invasion and metastasis in breast cancer cells. However, the mechanism that TGF-β functions by remains largely unknown. In the present study we report the mechanism of ERK1/2 dependant S100A4 regulation by TGF-β and its possible role in TGF-β-mediated tumour invasion in vitro. MATERIALS AND METHODS: Small interfering RNA targeting TGF-β1 (TGF-β1 siRNA) were stably transfected into the breast cancer cell line MDA231. The TGF-β1 siRNA/9MDA231 cells were then treated with TGF-β1 (5 ng/ml) or treated with PD98059 (25 μM) or transfected into S100A4 siRNA before TGF-β1 treatment. The cells were used in several in vitro analyses, including migration, invasion, angiogenesis, 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. In vitro angiogenesis studies using conditioned medium in HMEC-1 cells. RESULTS: Inhibition of TGF-β1 expression by TGF-β1 siRNA transfection in MDA231 cells showed significant decrease migration, invasion and angiogenesis in vitro. TGF-β1 siRNA/MDA231 cells treated with 5 ng/ml TGF-β1 for 24 hs restored the invasive ability of TGF-β1 siRNA/MDA231 cells. TGF-β1 treatment could not increase migration, invasion and angiogenesis in TGF-β1 siRNA/MDA231 cells when treated with 25 μM PD98059 or transfected with S100A4 siRNA before TGF-β1 treatment. Analysis of TGF-β1 signaling pathways showed a decrease in p-ERK1/2 activation and an decrease in S100A4 expression. Interestingly, TGF-β1 regulated S100A4 via ERK1/2 signalling. CONCLUSIONS: Our findings showed that blocking TGF-β inhibits breast cancer cell invasiveness, migration and angiogenesis via ERK/S100A4 signalling. Therapies targeting the TGF-β signaling pathway may be more effective to prevent progression in breast cancer.