The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) is abundant in decidua but its role in placental development is unclear. We hypothesized that decidual 1,25(OH)2D promotes placentation in early-pregnancy via actions on trophoblastic cell invasion. Using trophoblastic JEG3, BeWo and HTR8 cells we showed that 1,25(OH)2D has no effect on trophoblastic cell proliferation and migration in plasticware culture. However, in Matrigel transwell cultures, 1,25(OH)2D potently promoted trophoblastic cell invasion. Vitamin D system analysis showed complete absence of the nuclear vitamin D receptor (VDR) in plasticware culture of JEG3, BeWo and HTR8 cells, whilst TPC cells showed strong nuclear VDR and potent 1,25(OH)2D-mediated induction of VDR target genes such as CYP24A1. Conversely, on Matrigel, trophoblastic cells showed abundant expression of VDR, with stronger nuclear localization in the presence of 1,25(OH)2D, whilst TPC showed no VDR. Notably, all cells showed intracellular expression of the serum vitamin D binding protein (DBP) in Matrigel but not plastic culture. Intracellular expression of DBP appeared to be due to uptake of exogenous DBP, with serum-free cultures showing no intracellular DBP. Trophoblastic and TPC cells showed non-genomic response to 1,25(OH)2D through induction and nuclear localization of phosphoERK1/2 (pERK1/2), and inhibition of ERK1/2 blocked DBP uptake and trophoblast Matrigel invasion. Likewise, low serum/low DBP culture dramatically suppressed trophoblastic matrix invasion. These data suggest VDR and DBP cooperate to promote decidual invasion by trophoblastic cells, with 1,25(OH)2D enhancing cellular uptake of serum DBP by pERK-mediated signaling. We, therefore, propose that healthy placentation requires the actions of both 1,25(OH)2D and its serum carrier protein, with maternal levels of DBP and its uptake by trophoblastic cells being a new important consideration in the overall impact of vitamin D on pregnancy success.