The loss of epithelial polarity is thought to play an important role during mammary tumor progression. Using a unique transgenic mouse model of ErbB2-induced mammary tumorigenesis, we demonstrated previously that amplification of ErbB2 is frequently accompanied by loss of the 14-3-3sigma gene. Here, we demonstrate that ectopic expression of 14-3-3sigma results in restoration of epithelial polarity in ErbB2-transformed mammary tumor cells. We further demonstrate that targeted deletion of 14-3-3sigma within primary mammary epithelial cells increases their proliferative capacity and adversely affects their ability to form polarized structures. Finally, we show that 14-3-3sigma can specifically form complexes with Par3, a protein that is essential for the maintenance of a polarized epithelial state. Taken together, these observations suggest that 14-3-3sigma plays a critical role in retaining epithelial polarity.