Recombinant murine S100A4 promotes growth and invasion of transformed mammary epithelial cells through an RAGE/ERK pathway. S100A4 is a calcium-binding protein known for its ability to promote cancer invasion, metastasis and angiogenesis. Recent studies suggest S100A4 also promotes breast cancer cell proliferation. However, the mechanism by which S100A4 promotes proliferation and/or invasion remains unknown. In this study, we show that S100A4 mRNA and protein are increased in multiple breast cancer cell lines and tissues when compared to normal mammary cells. To investigate whether S100A4 promotes growth and invasion in transformed breast epithelial cells, S100A4 was expressed in the non-transformed mammary epithelial MCF10A cell line and the invasive breast carcinoma MDA-MB-231 cell line using a replication-defective retroviral vector system. Upon infection, the endogenous S100A4 mRNA and protein levels increased several-fold over endogenous levels in both cell lines. In addition, the expression of exogenous S100A4 induced expression of the breast cancer-associated proteins epidermal growth factor receptor (EGFR) and matrix metalloproteinases (MMP)-1, -2, and -9. Conversely, we also found that knockdown of S100A4 reduced the expression of EGFR and MMPs and delayed cell growth. Furthermore, S100A4 induced cell invasion and its expression correlated with expression of EGFR, p-EGFR, p-ERK, and the invasion marker MMP-9. To determine whether S100A4 regulates EGFR and MMP expression through S100A4/RAGE signaling, we used siRNA to knock down endogenous S100A4 and recombinant soluble RAGE protein to block the interaction between S100A4 and its receptor. Both of these approaches inhibited EGFR and MMP expression. The S100A4 induced growth promoting activity was abrogated when S100A4 was knocked down with siRNA and suppressed when endogenous S100A4 was blocked by recombinant RAGE protein. Our findings suggest that S100A4 promotes the invasive phenotype of breast epithelial cells through an S100A4/RAGE/ERK pathway. Therefore, our results provide evidence to support the hypothesis that breast cancers expressing high levels of S100A4 will have increased growth and invasion.