Background: Lactoferrin (Lf) is a multifunctional protein and one of the most abundant proteins in human milk. Various factors may affect its concentration in human milk, such as stage of lactation, ethnicity, and diet. Objectives: The objectives of the present study were to examine the dynamic change in milk Lf throughout the course of lactation and explore factors associated with milk Lf concentrations in various Chinese populations. Methods: This investigation was a part of a large cross-sectional study conducted in 11 provinces/autonomous regions/municipalities (Beijing, Gansu, Guangdong, Guangxi, Heilongjiang, Inner Mongolia, Shandong, Shanghai, Xinjiang, Yunnan, and Zhejiang) across China between 2011 and 2013. Lactating women (n
= 6481) within 0–330 days postpartum were recruited in the original study. A sub-sample of 824 women was randomly selected, and milk Lf concentrations were determined by UPLC/MS. Results: The Lf concentration in milk from women delivering at term was 3.16 g/L, 1.73 g/L and 0.90 g/L for colostrum, transitional milk, and mature milk, respectively. Lf concentrations differed significantly between stages of lactation (colostrum vs. transitional milk, colostrum vs. mature milk, transitional milk vs. mature milk, all p
< 0.001). Maternal BMI, age, mode of delivery, parturition, protein intake, and serum albumin concentration were not correlated with milk Lf concentration. However, milk Lf concentrations varied among different geographical regions (Guangdong (1.91 g/L) vs. Heilongjiang (1.44 g/L), p
= 0.037; Guangdong (1.91 g/L) vs. Gansu (1.43 g/L), p
= 0.041) and ethnicities (Dai (1.80 g/L) vs. Tibetan (0.99 g/L), p
= 0.007; Han (1.62 g/L) vs. Tibetan (0.99 g/L), p
= 0.002) in China. Conclusions: The concentration of Lf in human milk changes dynamically throughout lactation. Few maternal characteristics affect the milk Lf concentration, but it varies across different geographical regions and ethnicities in China.