To determine the contents of B-vitamins in human milk in China, we analyzed 1778 human milk samples from the sample bank of the National High Technique R & D Program (863 Projects) which was a cross-sectional survey and covered 6419 human milk samples from healthy lactating mothers who were at different stages of lactation (0-330 days postpartum) in 11 provinces of China. The contents of free forms of six B-vitamins in these human milk samples were analyzed by using UPLC-MS/MS. The median concentrations of free form of 6 B-vitamins in colostrums, transitional milk, 15-180 d mature milk and 181-330 d mature milk were respectively as follows: thiamin 5.0 µg/L, 6.7 µg/L, 21.1 µg/L and 40.7 µg/L; riboflavin 29.3 µg/L, 40.6 µg/L, 33.6 µg/L and 29.6 µg/L; niacin 470.7 µg/L, 661.3 µg/L, 687.0 µg/L and 571.3 µg/L; vitamin B-6 4.6 µg/L, 16.1 µg/L, 62.7 µg/L and 80.7 µg/L; flavin adenine dinucleotide (FAD) 808.7 µg/L, 1162.8 µg/L, 1023.9 µg/L and 1057.2 µg/L; pantothenic acid 1770.9 µg/L, 2626.8 µg/L, 2213.0 µg/L and 1895.5 µg/L. The contents of 6 B-vitamins varied significantly among the different lactation stages and different areas (coastal area vs inland area, rural area vs urban area). The present study indicated that the concentrations of B-vitamins in colostrum were generally much lower than those in transitional milk and mature milk. Further studies are warranted for their roles and significance on B-vitamins in colostrum in nutrition and metabolism of neonates.