Downregulation of CPEB1, a sequence-specific RNA-binding protein, in a mouse mammary epithelial cell line (CID-9) causes epithelial-to-mesenchymal transition (EMT), based on several criteria. First, CPEB1 knockdown decreases protein levels of E-cadherin and β-catenin but increases those of vimentin and Twist1. Second, the motility of CPEB1-depleted cells is increased. Third, CID-9 cells normally form growth-arrested, polarized and three-dimensional acini upon culture in extracellular matrix, but CPEB1-deficient CID-9 cells form nonpolarized proliferating colonies lacking a central cavity. CPEB1 downregulates Twist1 expression by binding to its mRNA, shortening its poly(A) tract and repressing its translation. CID-9 cultures contain both myoepithelial and luminal epithelial cells. CPEB1 increases during CID-9 cell differentiation, is predominantly expressed in myoepithelial cells, and its knockdown prevents expression of the myoepithelial marker p63. CPEB1 is present in proliferating subpopulations of pure luminal epithelial cells (SCp2) and myoepithelial cells (SCg6), but its depletion increases Twist1 only in SCg6 cells and fails to downregulate E-cadherin in SCp2 cells. We propose that myoepithelial cells prevent EMT by influencing the polarity and proliferation of luminal epithelial cells in a mechanism that requires translational silencing of myoepithelial Twist1 by CPEB1.