The epithelial–mesenchymal transition (EMT) is a process in which polarized epithelial cells are converted into motile mesenchymal cells. During cancer development, EMT is conducive to tumor dissemination and metastatic spread. While overexpression of metadherin (MTDH) in breast cancer cell lines and tissues has been found to be associated with aggressive tumor behavior, its precise role in invasion and metastasis is largely unknown. Here we report that MTDH overexpression could significantly enhance the invasion and migration of breast cancer cells by inducing EMT. Metadherin overexpression led to upregulation of mesenchymal marker fibronectin, downregulation of epithelial marker E-cadherin, and the nuclear accumulation of beta-catenin. Also, transcription factors Snail and Slug were upregulated in breast cancer cells overexpressing MTDH. Overexpression of MTDH enhanced the invasiveness and migration ability of breast cancer cells in vitro. In addition, overexpression of MTDH led to increased acquisition of CD44+/CD24−/low markers that are characteristic of breast cancer stem cells. We also showed that NF-kappa was involved in the expression of EMT-related markers. Taken together, our results suggest that MTDH could promote EMT in breast cancer cells in driving the progression of their aggressive behavior. (Cancer Sci 2011; 102: 1151–1157)