BACKGROUND: Adequate B-vitamins concentrations in human milk are considered to be a prerequisite for healthy development of infants in early life. This study aims to determine the concentrations of B-vitamins in human milk from Chinese women and the relationships between their concentrations and different geographical origin, lactation stages, socioeconomic characteristics, and dietary intake. METHODS: Human milk was obtained from 443 healthy lactating women from Beijing (n = 150), Suzhou (n = 146), and Guangzhou (n = 147) cities. Thiamine, riboflavin, vitamin B3 (nicotinamide and nicotinic acid), and vitamin B6 (pyridoxal, pyridoxine, and pyridoxamine) in human milk were analyzed by high performance liquid chromatography-tandem mass spectrometry. Pantothenic acid, biotin, and folates in human milk were analyzed by microbiological assay. The information from one 24-h dietary recall and socioeconomic characteristics were collected by interview and structured questionnaire, respectively. RESULTS: B-vitamins concentrations in human milk varied greatly among individuals. The median concentrations of B-vitamins of postpartum 5-11 d, 12-30 d, 31-60 d, 61-120 d, and 121-240 d were respectively as follows: thiamine 3.13, 5.07, 4.28, 5.65, 6.28 (µg/100 g); riboflavin 20.8, 20.2, 11.9, 13.6, 15.6 (µg/100 g); vitamin B3 194.0, 300.0, 261.0, 212.5, 218.0 (µg/100 g); pantothenic acid 236.5, 291.0, 254.0, 179.0, 189.0 (µg/100 g); vitamin B6 6.34, 7.58, 8.60, 9.34, 10.20 (µg/100 g); biotin 0.462, 0.834, 0.606, 0.523, 0.464 (µg/100 g); folates 0.730, 2.390, 2.440, 2.420, 2.330 (µg/100 g). The levels of B-vitamins presented regional differences and varied significantly among different lactation stages. The inversely associations of thiamine, vitamin B6, and folates with maternal BMI were found in multivariate analyses (p < 0.05), as well as higher pantothenic acid, folates, and biotin concentrations in lactating women with supplement intake when compared with those without (p < 0.05). Riboflavin concentrations associated with regular exercise was found in multivariate analyses (p < 0.05). CONCLUSIONS: The present study indicated regional and socioeconomic factors, lactation stage, and supplement intake may influence B-vitamins concentrations of human milk in healthy Chinese mothers. Further studies on accurate and complete analysis of all vitamin forms are crucial for giving a more comprehensive understanding of vitamin status in human milk. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01971671. Registered 13 October 2013.