Micronutrient status may be a contributing factor to the development of childhood obesity in many industrializing countries passing the nutritional transition. The few studies investigating associations between serum concentrations of vitamin B and intake of B vitamins with adiposity, however, have reported inconsistent findings. The aim of the study was to examine associations between serum vitamin B-12 and folate concentrations and intakes of B vitamins with body fat by using data on 1131 Mexican American children 8-15 y of age included in NHANES 2001-2004. Children's body mass index (BMI), trunk fat mass (TrFM), and total body fat mass (TBFM) were used as body adiposity (BA) measures. Serum concentrations of folate and vitamin B-12 were measured in blood samples collected from children. Intake of B vitamins was collected according to 24-h dietary recall. Associations of BA with serum concentrations of vitamin B-12 and folate and intake of B vitamins were determined by using linear and multinomial regression models. Serum concentrations of vitamin B-12 and folate were inversely associated with BMI (β: -2.68, P < 0.01; β = -1.33, P < 0.01), TrFM (β:-3.32, P < 0.01; β: -0.14, P < 0.05), and TBFM (β:-1.93, P < 0.01; β: -3.19; P < 0.01). Higher serum concentrations of vitamin B-12 were associated with a reduced risk of obesity (OR: 0.48; 95% CI: 0.31, 0.77; P < 0.001). Thiamin and riboflavin intakes were inversely associated with BMI (β:-1.35, P < 0.05; β: -1.11, P < 0.05) and TrFM (β:-1.26, P < 0.05; β: -1.37, P < 0.05). Similar inverse associations between BA and status of both vitamin B-12 and folate and intake of thiamin and riboflavin suggest that these micronutrients may play a role in adipogenesis and risk of childhood obesity.