The aim of this study was to determine the prevalence of 25-hydroxyvitamin D (25(OH)D) deficiency in a hospital-based population of both native Dutch and non-Western immigrants and to investigate the influence of immigrant status on the prevalence of vitamin D deficiency. A cross-sectional survey was conducted among 132 patients (1-18 years of age) visiting the paediatric outpatient department. Serum levels of 25(OH)D were measured using high-performance liquid chromatography. Cut-off levels of 30 and 50 nmol/l for serum 25(OH)D were evaluated. One third of the patients had serum 25(OH)D levels below 30 nmol/l, and half of the study population had serum levels below 50 nmol/l. Non-Western immigrants had an increased risk for vitamin D deficiency compared to their native Dutch peers [25(OH)D of <30 nmol/l, p = 0.03, odds ratio (OR) 3.87 (95 % confidence interval (CI) 1.13-13.29); 25(OH)D of <50 nmol/l, p = 0.02, OR 3.57 (95 % CI 1.26-10.14)] with the highest risk for first-generation non-Western immigrants. CONCLUSIONS Vitamin D deficiency in the paediatric population is still a matter of concern in the Netherlands, in particular among first-generation non-Western immigrants. We therefore strongly recommend vitamin D supplementation for all non-Western immigrants, regardless of age, skin type or season. Health-care staff who work with non-Western immigrants should be aware of the prevalence and implications of vitamin D deficiency.