Ankyloglossia (tongue tie) is a well-recognized cause of breastfeeding difficulties and, if untreated, can cause maternal nipple pain and trauma, ineffective feeding, and poor infant weight gain. In some cases, this condition will result in a downregulation of the maternal milk supply. Milk-production measurements (24-hour) for a breastfeeding infant with ankyloglossia revealed the ineffective feeding of the infant (78 mL/24 hours), and a low milk supply (350 mL/24 hours) was diagnosed. Appropriate management increased milk supply (1254 mL/24 hours) but not infant milk intake (190 mL/24 hours). Test weighing convincingly revealed the efficacy of frenotomy, increasing breastfeeding milk transfer from 190 to 810 mL/24 hours. Postfrenotomy, breastfeeding almost completely replaced bottle-feeding of expressed breast milk. This case study confirms that ankyloglossia may reduce maternal milk supply and that frenotomy can improve milk removal by the infant. Milk-production measurements (24-hour) provided the evidence to confirm these findings.