Vitamin D deficiency during pregnancy is a public health problem in Pakistan, and prevalent among most women of reproductive age in the country. Vitamin D supplementation during pregnancy is suggested to prevent adverse pregnancy outcomes and prevent vitamin D deficiency in both the mother and her newborn. However, there remains uncertainty surrounding the appropriate and safe dose for vitamin D supplementation in pregnant women who experience deficiency. We conducted a double-blinded, randomized, controlled, dose-comparison trial aimed to evaluate the relationship between different doses of vitamin D supplementation during pregnancy and pregnancy outcomes among women in a maternity hospital of the Aga Khan University (AKU) in Karachi, Pakistan. Pregnant women (n=350) in their first trimester were recruited and randomized to three treatment groups of vitamin D supplementation: 4,000 IU/day (Group A; n=120), 2,000 IU/day (Group B; n=115), or 400 IU/day (Group C – control; n=115). Deficiency in serum vitamin D (serum 25OHD <20ng/mL) at baseline was prevalent in more than 95% of women in each group. Participants in group A had the lowest vitamin D deficiency at endline and in newborns (endline: 75.9%; neonatal: 64.9%), followed by group B (endline: 84.9%; neonatal: 73.7%) and then the control group (endline: 90.2%; neonatal: 91.8%). Vitamin D deficiency was significantly lower in group A than group C (p=0.006) for women at endline, and lower in both group A and group B than the control group (p=0.001) in neonates. There were no adverse events attributable to vitamin D supplementation in all groups. Our study concluded that vitamin D supplementation with 4000 IU/day is safe and effective in reducing the risk of maternal and neonatal vitamin D deficiency.