Abstract Background We evaluated population characteristics of serum 25-hydroxyvitamin D (25(OH)D) levels and determined the influence of age, gender and season in an extensive dataset. Materials and methods Laboratory results of 103,509 adults and 19,186 children were retrospectively evaluated. Study group was classified regarding ages as; <40, 40–50, 50–60 and >60 years for adults and 0–1, 1–12 months,1–3, 4–6, 7–9, 10–12, 13–15 and 16–18 years for children. Seasonal values were also determined. Levels were measured by Architect i1000 SR (Abbott Diagnostics, USA). Results The median (2.5–97.5 percentiles) of 25(OH)D levels were 38.75 (9.5–158.25) nmol/L for adults and 43.25 (11.25–125.5) nmol/L for children. There were significant gender differences for both adults and children. Values differed significantly among age subgroups (p’s < 0.01). A total of 63% of adults and 59.5% of children had 25(OH)D levels below 50 nmol/L (p < 0.001). 25(OH)D levels were significantly lower in the winter compared with summer (p’s < 0.001). Even levels in summer were moderate deficient for all group. Conclusion The rate of 25(OH)D deficiency was remarkable during the whole year. This will provide large-scale data about 25(OH)D status in Turkish people and may contribute to the prevention and treatment of this condition for better healthcare outcomes.