Development of three-dimensional (3D) cultures that mimic in vivo tissue organization has a pivotal role in the investigation of the involvement of cell adhesion and polarity genes in the pathogenesis of epithelial cancers. Here we describe a novel 3D culture model with primary mouse endometrial epithelial cells. In this model, isolated endometrial epithelial cells develop single-lumened, polarized glandular structures resembling those observed in endometrial tissue. Our in vitro 3D culture model of endometrial glands requires the use of serum-free defined medium with only epidermal growth factor and insulin as growth supplements and 3% Matrigel as reconstituted extracellular matrix. Under these culture conditions, glands of epithelial cells displaying typical apicobasal polarity and proper positioning of tight and adherent junctions are formed by hollowing as early as 7 to 8 days in culture. Addition of the phosphatidylinositol 3-kinase inhibitor LY294002 completely inhibits bromodeoxyuridine incorporation and cyclinD1 expression, confirming that in vitro growth of endometrial glands depends on phosphatidylinositol 3-kinase/Akt signaling. To prove that our culture method is a good model to study endometrial carcinogenesis, we knocked down E-cadherin or phosphatase and tensin homolog expression by lentivirus-delivered short hairpin RNAs. Down-regulation of E-cadherin resulted in complete loss of epithelial cell polarity and glandular formation, whereas phosphatase and tensin homolog down-regulation resulted in increased proliferation of glandular epithelial cells. These properties indicate that our 3D culture model is suitable to study the effect of growth factors, drugs, and gene alterations in endometrial carcinogenesis and to study normal endometrial biology/physiology.