Vitamin D is important for calcium absorption and skeletal growth. Vitamin D insufficiency (VDI) is a prevalent health problem in children. A study was performed to determine the prevalence of VDI in healthy children living in Tehran, Iran. In a cross-sectional study, 963 students (424 boys and 539 girls) aged 7–18 years were selected by random sampling. Serum 25-hydroxyvitamin D (25-OHD), calcium, alkaline phosphatase and phosphorus were measured. VDI was defined as serum 25-OHD <20 ng/ml. Prevalence of VDI was 53.6% in girls and 11.3% in boys. VDI in female students was about five times more common than males (p < 0.000001). VDI in children and adolescent girls is a health problem not only for these age-groups but also for the next generation to come. Encouraging girls to have more sun exposure, fortification of foods and prescription of supplemental vitamin D are recommended.