Endocrine hormones of the placenta are essential for the maintenance of pregnancy, and disturbances in trophoblast integrity are associated with pregnancy complications. Here, we combine human trophoblast cell cultures, hormone assays, antibody-based detection methods and high-resolution mass spectrometry analyses to assess changes in cellular processes during syncytialisation. We monitored the changes in protein expression profiles during trophoblast syncytialization in an unbiased manner and show that the expression of numerous proteins is strongly altered. Among them are enzymes of the glucocorticoid and sex hormones synthesis pathways such as CYP19A1, CYP11A1, FDX1, HSD11B2 and HSD17B1, whose expression is strongly induced by syncytialization. The production of β-hCG, progesterone and estradiol increases several-fold during syncytialization, while the secretion and synthesis of ß-hCG and the expression of several syncytiotrophoblast markers show a clear calcium dependence