Introduction World-wide vitamin D (vitD) deficiency in pregnancy remains a frequent obstetrical challenge known to be associated with an increased risk of placenta-related complications. While some small-scale trials have indicated beneficial effects of high dose vitD supplementation on various pregnancy outcomes, research on vitD effects on placental biology and the placental response to increased supplementation are limited. Aim The GRAVITD trial explores vitD status among Danish pregnant women and tests the currently recommended dose of vitD (10 µg) against high-dose supplementation (90 µg). This trial was designed with sufficient power to determine if there is a beneficial effect on pregnancy outcomes such as preeclampsia, fetal growth retardation and gestational diabetes as well as examining the effects of increased vitD supplementation on the placenta. Methods A total of 2000 pregnant women were enrolled at the end of first trimester and randomized in a 1:1 ratio to 10 µg or 90 µg vitD3. First trimester blood samples were analyzed for 25-hydroxyvitamin D (25(OH)D) from 1,971 women. Of these, 1,854 completed the trial. Umbilical cord blood from 475 neonates were analyzed for 25(OH)D. Placental samples were analyzed by qPCR for a subset of the participants (n=118) to investigate effects on vitD transporters (Cubilin; CUBN and megalin; LRP2) and the vitD sensitivity i.e., the expression of CYP24A1, CYP27B1 and VDR. In addition, next-generation RNA sequencing (NGS) was performed on a subset of placental samples (n=70) to examine functional effects of the two vitD doses. Results At the end of first trimester, 42% of pregnant women displayed 25(OH)D concentrations below the current recommendation for pregnancy (75 nmol/L). Increased vitD supplementation significantly improved vitD status among the pregnant women with 97.5% achieving 25(OH)D ≥75 nmol/L in the 90 µg group compared to 76.5% in the 10 µg group. Moreover, analysis of umbilical cord blood revealed that 51% neonates born in the 10 µg group were vitD deficient (25(OH)D <50 nmol/L) in contrast to only 11% in the 90 µg group. High dose vitD supplementation did not affect the placental vitD uptake or -metabolism remarkably. However, higher pre-pregnancy BMI led to differential expression of CUBN, CYP24A1 and CYP27B1 as a response to the allocated vitD dose. NGS data showed that signaling pathways related to cell adhesion, immune function, and neurodevelopment were affected by the given vitD dose. Perspectives Results on clinical outcomes are still pending but preliminary analysis show a beneficial effect on preterm birth.