Calcitriol levels increase during pregnancy, contributing to the hormonal and immunological balance, but its deficiency has been associated with problems during this period. Meanwhile, transforming growth factors-β (TGF-βs) play an important role in the maintenance of fetal-maternal immune tolerance; however, exacerbated concentrations of this growth factor are associated with complicated pregnancies. Therefore, we studied the effects of calcitriol on TGF-βs and their receptors in trophoblast cells. Term placentas from uncomplicated pregnancies after cesarean sections were used for cell cultures. Basal gene expression and the effect of calcitriol upon TGF-β1, TGF-β2, TGF-β3, and their receptors TGF-βR1 and TGF-βR2 were assessed using real-time PCR from trophoblast cells. The presence of TGF-β1, 2, 3, and TGF-βR1 were evaluated by immunofluorescence, and the protein abundance and secretion of TGF-β1 were assessed by Western blot and ELISA, respectively. Basal gene expression of TGF-β1 in trophoblast from term placentas was higher than TGF-β2 and TGF-β3, while TGF-βR2 was higher than TGF-βR1. The presence and cellular localization of TGF-β1, 2, 3, and TGF-βR1 were detected in the cytoplasm of syncytiotrophoblast, with TGF-β1 showing the highest intensity. Calcitriol significantly inhibited gene expression of TGF-β1, TGF-β2, and TGF-βR1. Likewise, calcitriol decreased the secretion and abundance of TGF-β1. In conclusion, results indicate that calcitriol is a regulator of TGF-βs in cultured trophoblast cells from term placentas and therefore may be an important player in the development of healthy pregnancies.