Objective: Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. Approach and Results: We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1α, 25-(OH)2-D in non-pregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1α, 25-(OH)₂-D compared to normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not further increase the plasma levels of 25-OH-D but it significantly increased those of the active metabolite, 1α, 25-(OH)₂-D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D activating enzyme, CYP27b1, and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung with opposite effects on placental VDR. Conclusions: Ovine pregnancy is a model of increased vitamin D status and long-term hypoxia further improves vitamin D status due to pregnancy- and hypoxia-specific regulation of VDR and metabolic enzymes.