MiR-21 is known to play an important role in the development and progression, including migration and invasion, of many malignancies including breast cancer. Accumulating evidence suggest that the induction of epithelial-mesenchymal transition (EMT) phenotype and acquisition of cancer stem cell (CSC) characteristics are highly interrelated, and contribute to tumorigenesis, tumor progression, metastasis, and relapse. The molecular mechanisms underlying EMT and CSC characteristics during miR-21 contributes to cell migration and invasion of breast cancer are poorly understood. Therefore, we established miR-21 re-expressing breast cancer MCF-7 (MCF-7/miR-21) cells, which showed increasing cell growth, migration and invasion, self-renewal and clonogenicity. Our data showed that re-expression of miR-21 induced the acquisition of EMT phenotype by activation of mesenchymal cell markers (N-cadherin, Vimentin, α-SMA) and inhibition of epithelial cell marker (E-cadherin) in MCF-7/miR-21 cells, which consistent with increased cell subpopulation expressing CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and the capacity of sphereforming (mammospheres). Our results demonstrated that re-expression of miR-21 is responsible for migration and invasion by activating the EMT process and enhancing the characteristics of CSCs in MCF-7 cells.