In an inducible oncogenesis model, the miR-200 family is inhibited during CSC formation but not transformation, and inhibition of miR-200b increases CSC formation. Interestingly, miR-200b directly targets Suz12, a subunit of a polycomb repressor complex (PRC2). Loss of miR-200 during CSC formation increases Suz12 expression, Suz12 binding, H3-K27 trimethylation, and Polycomb-mediated repression of the E-cadherin gene. miR-200b expression or Suz12 depletion blocks the formation and maintenance of mammospheres, and in combination with chemotherapy suppresses tumor growth and prolongs remission in mouse xenografts. Conversely, ectopic expression of Suz12 in transformed cells is sufficient to generate CSCs. The miR-200b-Suz12-cadherin pathway is important for CSC growth and invasive ability in genetically distinct breast cancer cells, and its transcriptional signature is observed in metastatic breast tumors. The interaction between miR-200 and Suz12 is highly conserved, suggesting that it represents an ancient regulatory mechanism to control the growth and function of stem cells.