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Abstract Introduction In utero fetal development and fetal programming for adulthood life are strongly associated with maternal-to-fetal transfer of nutrient and other substances. Gestational diabetes mellitus (GDM) is a major problem and associated with abnormal fetal development, but the mechanisms underlying glucose transport across the placenta barrier (PB) are not completely understood. Methods We developed a placenta simulator that can mimic feto-maternal blood circulations along with real transfer across the in vitro biological model of the PB, which is made of a co-culture of endothelial cells (EC) and trophoblast cells (TC) on both sides of a denuded amniotic membrane (AM). Maternal-to-fetal transfer of glucose was monitored over 24 h. Results The AM is highly permeable to glucose compared to the cellular structures and can serve as a substrate for the co-culture model. The transfer characteristics for glucose are independent of its initial concentration in the maternal compartment, but strongly dependent on the cellular components of the PB. The EC are more resistive to glucose transfer than the TC. The in vitro PB model is the most resistive to glucose transfer. Discussion and conclusion The good correlation between the present in vitro results with existing in vivo data demonstrated the potential of this new approach, which can be extended to study various aspects of transplacental transfer, including medications, relevant to GDM or any problem related to in utero programing.