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Choline is an essential micronutrient with critical roles in human development. Infants require large amounts of choline throughout the first year of life to support organ growth and membrane biosynthesis. Human milk is rich in choline and provides the infant with about three times more choline (per kilogram of body weight) than what an adult would consume on a normal diet. However, several factors can impact milk choline supply including maternal dietary choline intake, genetic variants, and stage of lactation. Choline uptake by the mammary epithelium occurs by a saturable energy-dependent transport system, although non-saturable transport can occur with a higher maternal choline supply. Other choline metabolites are derived primarily through synthesis or degradative pathways within the mammary epithelium. Efflux of choline metabolites from the mammary epithelium depends upon the choline form and occurs via exocytosis or as a component of the milk fat globule. The choline adequate intake for lactating women is 550 mg/d which is almost double the amount of choline typically consumed by a woman of childbearing age residing in the United States. As choline is absent from most prenatal vitamins, efforts to increase dietary choline consumption throughout lactation are needed to optimize maternal and infant well-being.