Vitamin D deficiency is prevalent in pregnant women and associated with adverse pregnancy outcomes, including pre-eclampsia. Active vitamin-D (1,25(OH)2D3) exerts important non-classical immune-regulatory effects, and the maternal placenta (decidua) appears a key target. Uterine natural killer cells (uNKs) are the most prominent decidual immune cell during early pregnancy. Given their critical role in fetal implantation and placentation, we hypothesised uNKs are a local source and pivotal target for 1,25(OH)2D3. CD56+ uNK and peripheral NKs (pNKs) were isolated from paired-decidua and blood from pregnant women undergoing 1st trimester surgical termination. NKs were cultured ± cytokine-stimulation (CK) ±1,25(OH)2D3 (10 nM). uNK and pNKs express the vitamin D-activation enzyme CYP27B1 and receptor (VDR), with both up-regulated by CK (P<0.05). Addition of 1,25(OH)2D3 attenuated this response (P<0.05), with concomitant up-regulation of CYP24A1. RNA sequence analysis (RNAseq) was performed for unbiased identification of 1,25(OH)2D3 targets using FACS sorted uNK and pNKs cultured with CK ±1,25(OH)2D3. The transcriptional patterns of NKs are highly tissue-specific; overall 2286 genes (1098 up-, 1188 down-regulated) were differentially expressed (DE) (P≤0.05, fold-change −0.5 to +1.5) in CK uNK and pNKs. For uNKs, 66 genes were DE (46 up-, 20 down-regulated) with 1,25(OH)2D3 comparative to CK alone. For pNKs, 71 were DE with 1,25(OH)2D3 (38 up-, 33 down-regulated). Only TRIM35, which inhibits cell proliferation and is anti-tumorigenic, was up-regulated in both Unk (P=0.0006, fold-change −1.86) and pNKs (P=0.01, fold-change −1.50). A unique transcriptional profile was identified in uNKs, with 1,25(OH)2D3-mediated effects upon genes associated with metabolism, migration, adhesion and apoptosis. Notably, 1,25(OH)2D3 increased galectin-9 (P=0.01, fold-change 1.75), which drives pNK transformation towards a decidua-phenotype, and SERPINB1, potent granzyme inhibitor (P=0.002, fold-change 2.37). NKs contain a functional vitamin D metabolic system, which appears particularly sensitive to 1,25(OH)2D3 within the decidua. RNAseq revealed a unique repertoire of 1,25(OH)2D3 targets, which appear highly relevant to decidualisation and materno-fetal tolerance.