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 B 3 (nicotinamide and nicotinic acid), and vitamin B 6 (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. 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 B 6 , 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).