Vitamin D is supplied in the newborn diet in Canada and the United States via supplement drops or formula. Vitamin D pills are commonly used by pregnant and nursing women. Because little is known about the impact of vitamin D supplementation on infant gut microbiota, researchers conducted a study in 1,157 mother-infant pairs from the CHILD (Canadian Healthy Infant Longitudinal Development) Cohort Study from 2009 to 2012 to see if there was a link between maternal and infant vitamin D supplementation, infant gut microbiota composition, and Clostridioides difficile colonization. To examine relationships between vitamin D supplementation and C. difficile colonization or other gut microbiota, logistic and MaAsLin regression were used. A vitamin D supplement was given to 65%of infants. Infant vitamin D supplementation was linked to a reduced abundance of the Megamonas genus in the gut microbiota (q=0.01) in all newborns. Maternal prenatal supplementation was related to decreased abundance of Bilophila (q=0.01) and Lachnospiraceae (q=0.02) but increased abundance of Haemophilus (q=0.02) among those solely breastfed. Vitamin D supplementation had no effect on the microbiota composition of partly and non-breastfed newborns. After controlling for breastfeeding status and other variables, neither infant nor maternal vitamin D supplementation was linked to C. difficile colonization. However, baby colonization of C. difficile was reduced when mothers drank vitamin-D supplemented milk (adjusted OR: 0.40, 95% CI: 0.19–0.82).