Insufficient 25-hydroxyvitamin D [25(OH)D] levels are common amongst pregnant women. Poor maternal 25(OH)D levels are associated with reduced fetal growth, reduced offspring bone density and bone mineral content, and childhood obesity. This suggests that poor maternal vitamin D levels may set the offspring on a pathway of poor health. 25(OH)D is the primary circulating form of vitamin D, however it is converted to a more active form, 1,25-dihydroxyvitamin D [1,25(OH)2D] within the body. This study investigates whether the human placenta is a site of 25(OH)D metabolism, and what vitamin D metabolites are transferred from the placenta to the maternal and fetal circulations. Term human placentas (n=5) were collected within 30 min of delivery with ethical approval and informed consent. The metabolism of 13C-25(OH)D3 within placental tissue and transfer across the placenta was investigated using the whole placental cotelydon perfusion open loop methodology as adapted in our laboratory. Maternal and fetal circulation was re-established, and the maternal circulation was perfused with 30 nmol/l 13C-25(OH)D3 for a period of 5h. Perfusate was collected periodically from both the maternal and fetal vein. The presence of vitamin D metabolites in perfusate samples was identified and quantified using liquid chromatography mass spectroscopy. Following 5h of perfusion there is transfer of 24.03% maternal 13C-25(OH)D from the maternal circulation into the placenta and 36.72% of this (8.82% of maternal stock) is transferred into the fetal circulation. There is evidence of placental metabolism of 13C-25(OH)D, with transfer of 13C-24,25-dihydroxyvitamin D [24,25(OH)2D] but not 13C-epi25(OH)D into the maternal and fetal circulations. qPCR measurement of the vitamin D responsive gene CYP24A1 indicates increased expression in the placental tissue following 5h perfusion of 13C-25(OH)D3. This indicates placental metabolism of 13C-25(OH)D3 into the transcriptionally active 1,25(OH)2D. This study provides evidence that once within the placenta the 25(OH)D can be metabolised and the downstream products could have effects within the placenta itself or be transferred to both the fetal and maternal circulation. Insufficient placental transport and metabolism of maternal 25(OH)D may therefore limit the fetal supply of 25(OH)D and 1,25(OH)2D, impacting fetal development. Placental transfer of 25(OH)D metabolites to the maternal circulation may contribute to the altered vitamin D status that occurs to help adapt the maternal physiology to support pregnancy.