Human embryo implantation involves a complex network of molecular signaling that is modulated by endocrine and paracrine pathways. Here, we performed studies using a unique and recently developed three-dimensional (3D) implantation model, characterized by an endometrium-like 3D culture system and Jar cell-derived spheroids mimicking the embryo/trophoblast. The aims were to investigate the effects of 17β estradiol (E2) and medroxyprogesterone acetate (MPA) on (1) the interaction between epithelial and stromal cells, and (2) the attachment and invasion of trophoblast cells. We observed that epithelial and stromal cells in the 3D culture were ERα⁺, ERβ⁺, and PR⁺. Decidualization was confirmed by enhanced prolactin gene expression on day 7 of E2 plus MPA treatment. An effect of epithelial cells on the decidualization of stromal cells was indicated by significantly higher levels of prolactin mRNA expression in the 3D culture compared to stromal cells grown within the fibrin-agarose gel matrix. On the other hand, the relative gene expressions of E-cadherin and IL-1β in epithelial cells of the 3D culture under decidualization conditions significantly differed from those in epithelial cells grown over the fibrin-agarose gel matrix without stromal cells, pointing to regulation of epithelial cells by the stroma. The attachment rate of Jar spheroids to the 3D was significantly increased by E2 plus MPA treatment. Analyses of Z-stack confocal and stained optic microscopic images demonstrated that Jar spheroids breached the epithelial cell monolayer, invaded, and were embedded into the 3D matrix in response to decidualization signals. In summary, the newly bioengineered system provides a unique model for studying interactions between the different endometrial cell compartments, via soluble-paracrine signals as well as cell-to-cell interactions, and is a useful tool to study early embryonic implantation events.