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Objective: The aim of the study was to determine the effects of age, gender and season on vitamin D status in healthy urban population at reproductive age. Also, we investigated the distribution of population into different groups regarding 25(OH)D levels. Methods: Serum 25(OH)D levels of 21,317 participants: 5,364 men (25.1%) and 15,953 women (74.8%), aged between 18-45 years, applying to two medical centres for check-up located in the same city were retrospectively analyzed. Group I consisted of 14,720 participants (11,257 women and 3,463 men) in the first centre and Group II consisted of 6,597 participants (4,696 women and 1,901 men) in the second centre. Results: The mean 25(OH)D levels did not differ between women and men in both groups: 23.4 (SD = 14.4) and 23.1 (SD = 12.6) in Group I, and 22.6 (SD = 15.9) and 23.1 (SD = 14.3) in Group II, respectively, (p > 0.05). Similar trends exhibiting lower mean 25(OH)D levels at younger ages and higher levels at later ages were observed in both groups; a seasonal variation of 25(OH)D levels was observed in both genders with the highest levels in August and September and the lowest levels from February through April; percentages of women with 25(OH)D level of < 5 ng/ml were significantly higher than of men in Group I (1.4% vs. 0.2%, respectively, p < 0.001) and in Group II (4.1% vs. 1.1%, respectively, p < 0.001). Conclusion: There is a slight increase in serum 25(OH)D levels from 18 through 45 years of age in healthy population. The seasonal variation of 25(OH)D levels is prominent in both genders with men having slightly lower levels in some months of winter and higher levels in summer as compared to women. The prevalence of women having 25(OH)D levels less than 5 ng/ml is higher than that of men.