Background <p>Lactoferrin (Lf) is an important bioactive protein in human milk involved in diverse biological functions. Concentrations of milk Lf are tightly regulated during lactation. This study aims to investigate the longitudinal changes in human milk Lf concentrations during the first year postpartum and identify the factors associated with these changes.</p> Methods <p>This prospective cohort study recruited 160 dyads of healthy mothers and their full-term newborns in Wuqiang, Hebei province, China. Maternal and infant characteristics were collected using a structured questionnaire. Milk Lf concentrations were measured using an ELISA. A linear mixed model was employed to examine the longitudinal changes in Lf concentrations and identify associated factors.</p> Results <p>The mean milk Lf concentrations were 7.17 g/L, 4.02 g/L, 2.95 g/L, 2.19 g/L, and 2.43 g/L at 0–7 days, 30 days, 3 months, 6 months, and 12 months postpartum, respectively. The frequency of breastfeeding in the prior 24 h was negatively associated with Lf concentrations (<i>P</i> = 0.017). Prenatal mean corpuscular volume (MCV) status may influence the longitudinal changes in milk Lf (<i>P</i>_MCV*time = 0.011).</p> Conclusions <p>Milk Lf concentrations decrease during the first 6 months and increase from 6 to 12 months, with these changes potentially associated with breastfeeding frequency and prenatal MCV status.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>This study represents one of the largest cohort investigations of the dynamic changes and influencing factors of human milk Lf concentration in China.</p> </ItemContent> <ItemContent> <p>The study investigated the relationship between maternal red blood cell indexes and milk Lf concentrations.</p> </ItemContent> <ItemContent> <p>Milk Lf concentrations dynamically change throughout lactation, decreasing during the first 6 months and potentially increasing from 6 to 12 months.</p> </ItemContent> <ItemContent> <p>The concentrations and longitudinal changes of milk Lf were impacted by the frequency of breastfeeding and maternal mean corpuscular volume.</p> </ItemContent> </UnorderedList></p>