Sufficient serum concentrations of vitamin D are required to maintain bone health during growth. The aims of this study were to determine whether vitamin D deficiency is more prevalent among children and adolescents with obesity compared to their normal weight peers and to identify clinical and biochemical variables associated with vitamin D deficiency. One thousand four hundred and eighty-four children and adolescents with overweight/obesity and 2143 population-based controls were recruited from the Danish Childhood Obesity Biobank. Anthropometric variables and fasting concentrations of serum 25-hydroxy vitamin D (25-OH-D), plasma parathyroid hormone (PTH), calcium and phosphate were assessed at baseline. Vitamin D deficiency was defined as serum 25-OH-D concentrations <30 nmol/L. Linear and logistic regressions were used to identify variables associated with vitamin D deficiency. A total of 16.5% of the children and adolescents with obesity (body mass index [BMI] standard deviation score [SDS]>2.33) exhibited vitamin D deficiency, with an odds ratio (OR) 3.41 (confidence interval [CI]: 2.27-5.71; p<0.0001) for being vitamin D deficient compared to their normal weight peers. BMI-SDS was independently and inversely associated with serum 25-OH-D concentrations. Other independent risk factors for vitamin D deficiency were being older than 14 years (OR: 2.39; CI: 1.28-4.48; p=0.006), more than 4 daily hours of screen time (OR: 4.56; CI: 2.59-8.05; p<0.0001) and blood sample assessment during winter-spring (OR: 6.44; CI: 4.47-9.26; p<0.0001). Vitamin D deficiency was common among Danish children and adolescents with obesity. The degree of obesity was independently associated with lower serum 25-OH-D concentrations.