Prenatal vitamin use is recommended before and during pregnancies for normal fetal development. Prenatal vitamins do not have a standard formulation, but many contain calcium, folic acid, iodine, iron, omega-3 fatty acids, zinc, and vitamins A, B6, B12, and D, and usually they contain higher concentrations of folic acid and iron than regular multivitamins in the US Nutrient levels can impact epigenetic factors such as DNA methylation, but relationships between maternal prenatal vitamin use and DNA methylation have been relatively understudied. We examined use of prenatal vitamins in the first month of pregnancy in relation to cord blood and placenta DNA methylation in two prospective pregnancy cohorts: the Early Autism Risk Longitudinal Investigation (EARLI) and Markers of Autism Risk Learning Early Signs (MARBLES) studies. In placenta, prenatal vitamin intake was marginally associated with -0.52% (95% CI -1.04, 0.01) lower mean array-wide DNA methylation in EARLI, and associated with -0.60% (-1.08, -0.13) lower mean array-wide DNA methylation in MARBLES. There was little consistency in the associations between prenatal vitamin intake and single DNA methylation site effect estimates across cohorts and tissues, with only a few overlapping sites with correlated effect estimates. However, the single DNA methylation sites with p-value < 0.01 (EARLI cord nCpGs = 4068, EARLI placenta nCpGs = 3647, MARBLES cord nCpGs = 4068, MARBLES placenta nCpGs = 9563) were consistently enriched in neuronal developmental pathways. Together, our findings suggest that prenatal vitamin intake in the first month of pregnancy may be related to lower placental global DNA methylation and related to DNA methylation in brain-related pathways in both placenta and cord blood.