During histiotrophic nutrition of the embryo, maternal platelets may be the first circulating maternal cells that find their way into the placental intervillous space through narrow intertrophoblastic gaps within the plugs of spiral arteries. Activation of platelets at the maternal-fetal interface can influence trophoblast behavior and has been implicated in serious pregnancy pathologies. Here, we show that platelet-derived factors impaired expression and secretion of the human chorionic gonadotropin beta-subunit (βhCG) in human first trimester placental explants and the trophoblast cell line BeWo. Impaired βhCG synthesis was not the consequence of hampered morphological differentiation, as assessed by analysis of differentiation-associated genes and electron microscopy. Platelet-derived factors did not affect intracellular cAMP levels and phosphorylation of CREB, but activated Smad3 and its downstream-target plasminogen activator inhibitor (PAI)-1 in forskolin-induced BeWo cell differentiation. While TGF-β type I receptor inhibitor SB431542 did not restore impaired βhCG production in response to platelet-derived factors, Smad3 inhibitor SIS3 interfered with CREB activation, suggesting an interaction of cAMP/CREB and Smad3 signaling. Sequestration of transcription co-activators CBP/p300, known to bind both CREB and Smad3, may limit βhCG production, since CBP/p300 inhibitor C646 significantly restricted its forskolin-induced upregulation. In conclusion, our study suggests that degranulation of maternal platelets at the early maternal-fetal interface can impair placental βhCG production, without substantially affecting morphological and biochemical differentiation of villous trophoblasts. KEY MESSAGES: Maternal platelets can be detected on the surface of the placental villi and in intercellular gaps of trophoblast cell columns from gestational week 5 onwards. Platelet-derived factors impair hCG synthesis in human first trimester placenta. Platelet-derived factors activate Smad3 in trophoblasts. Smad3 inhibitor SIS3 interferes with forskolin-induced CREB signaling. Sequestration of CBP/p300 by activated Smad3 may limit placental hCG production.