Mammosphere culture has been used widely for the enrichment of mammary epithelial stem cells and breast cancer stem cells (CSCs). Epithelial-to-mesenchymal transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether mammosphere culture conditions per se induced EMT in the epithelial MCF-7 breast cancer cell line. MCF-7 cells were cultured as mammospheres for 5 weeks, with dispersal and reseeding at the end of each week. This mammosphere culture induced a complete EMT by 3 weeks. Return of the cells to standard adherent culture conditions in serum-supplemented media generated a cell population (called MCF-7(M) cells), which displays a stable mesenchymal and CSC-like CD(44+)/CD(24-/low) phenotype. EMT was accompanied by a stable, marked increase in EMT-associated transcription factors and mesenchymal markers, and a decrease in epithelial markers and estrogen receptor α (ERα). MCF-7(M) cells showed increased motility, proliferation and chemoresistance in vitro, and produced larger tumors in immunodeficient mice with or without estrogen supplementation. MicroRNA analysis showed suppression of miR-200c, miR-203, and miR-205; and increases in miR-222 and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERα in MCF-7(M) cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.