Cells cultured as monolayers proliferate well, but do not sustain their differentiation characteristics. Previous studies have investigated the interactions between cells and growth factors or cytokines by establishing either in vivo or in vitro three-dimensional (3D) cultures. Using porcine uterine epithelial cells and endometrial cells, the current study was designed to develop a 3D uterine culture system and investigate the response to hormone treatment. Formation of the 3D uterine model was similar to that of uterus from the group supplemented with calcium and magnesium, and the addition of these ions altered the spectrum of basement membrane degrading enzyme expression and activity. In particular, the epithelial cell junctions in the 3D model most closely resembled those of an actual uterus when the medium was supplemented with calcium and magnesium; the intercellular basement membrane structure was also tall under these conditions. The study confirmed that Casp-3 expression was lowest in the P4 (progesterone) treatment group, and this hormone was the most potent stimulus for formation of the endometrial cell layer. Therefore, the addition of calcium and magnesium plays an important role in the formation of a 3D uterine model, and the addition of P4 hormone mimics uterine thickening by stimulating growth of the epithelial cell layer.