Objective: To explore the relationships of serum 25-hydroxyvitamin D (25(OH)D) with obesity and metabolic parameters in US children.Design: Cross-sectional analysis. We evaluated the associations between serum 25(OH)D and multiple measurements of adiposity, serum lipid concentrations, fasting glucose and insulin resistance in children aged 6–18 years with adjustments for multiple covariates.Setting: The National Health and Nutrition Examination Survey, 2001–2006.Participants: A nationally representative sample of 6311 children and adolescents aged 6–18 years.Results: Among US children and adolescents, the prevalence of vitamin D deficiency has been especially high in older children, girls and the non-Hispanic Black population. Higher odds of obesity were found at a 25(OH)D concentration of 50 nmol/l under both criteria for obesity in children (OR = 3·27, P_trend ≤ 0.001). Moreover, increased odds of having abnormal HDL-cholesterol (OR = 1·71, P_trend ≤ 0.001) and impaired insulin resistance (OR = 4·15, P_trend ≤ 0·001) were found for children deficient in 25(OH)D compared with those with normal 25(OH)D concentrations. When the children and adolescents were stratified by gender, we found stronger associations between serum 25(OH)D concentration and both HDL-cholesterol and insulin resistance in girls. No association of 25(OH)D with any other metabolic parameter was found.Conclusions: Our results suggest a potential harmful association between low serum 25(OH)D concentration and the risk of obesity among children. However, the underlying mechanisms require further investigation.