We assessed the response of primary cultures of placental villous mononucleated trophoblasts and multinucleated syncytiotrophoblast to calcitriol, the most biologically active form of vitamin D. Whole-genome microarray data showed that calcitriol modulates the expression of many genes in trophoblasts within 6 hours of exposure and RT-qPCR revealed similar responses in cytotrophoblasts, syncytiotrophoblasts and villous explants. Both cytotrophoblasts and syncytiotrophoblasts expressed genes for the vitamin D receptor, for LRP2 and CUBN that mediate internalization of calcidiol, for CYP27B1 that encodes the enzyme that converts calcidiol into active calcitriol, and for CYP24A1 that encodes the enzyme that modifes calcitriol and calcidiol to inactive calcitetrol. Notably, we found an inverse effect of calcitriol on expression of CD14 and CD180/RP105, proteins that differentially regulate toll-like receptor 4-mediated immune responses. Supported by gene ontology analysis, we tested the hypothesis that CD14 and CD180 modulate the infammatory response of syncytiotrophoblast to bacterial lipopolysaccharide (LPS). These cells showed a robust response to a wide range of LPS concentrations, with induction of active NF-κB and increased secretion of IL-6 and IL-8. SiRNA-mediated knockdown of CD14 reduced the secretion of IL-6 and IL-8 in response to LPS. Collectively, our data showed that calcitriol has a rapid and widespread effect on villous trophoblast gene expression in general, and a specific effect on the innate immune response by syncytiotrophoblast.