The active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) is a potent immunomodulatory seco-steroid. We have demonstrated that several components of vitamin D metabolism and signaling are strongly expressed in human uterine decidua from first trimester pregnancies, suggesting that locally produced 1,25(OH)₂D₃ may exert immunosuppressive effects during early stages of gestation. To investigate this further, we used primary cultures of human decidual cells from first and third trimester pregnancies to demonstrate expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)₂D₃, 1alpha-hydroxylase (CYP27B1). Synthesis of 1,25(OH)₂D₃ was higher in first trimester decidual cells (41 ± 11.8 fmoles/h/mg protein) than in third trimester cells (8 ± 4.4 fmoles/h/mg protein; P < 0.05). Purification of decidual cells followed by quantitative RT-PCR analysis showed that CYP27B1 was expressed by both CD10 ^VE stromal-enriched and CD10^−VE stromal-depleted cells, with higher levels of mRNA in first trimester pregnancies. Expression of CYP27B1 correlated with TLR4 and IDO. Functional responses to 1,25(OH)₂D₃ were studied using CD56 ^ve natural killer (NK) cells isolated from first trimester decidua. Decidual NK cells treated with 1,25(OH)₂D₃ or precursor 25-hydroxyvitamin D₃ (25OHD₃) for 28 h showed decreased synthesis of cytokines, such as granulocyte-macrophage colony stimulating factor 2 (CSF2), tumor necrosis factor, and interleukin 6, but increased expression of mRNA for the antimicrobial peptide cathelicidin antimicrobial peptide. These data indicate that human decidual cells are able to synthesize active 1,25(OH)₂D₃, particularly in early gestation, and this may act in an autocrine/paracrine fashion to regulate both acquired and innate immune responses at the fetal-maternal interface.