Data from previous studies demonstrate the high frequency of deficiency and insufficiency of vitamin D in Ukraine, as in the world, which varies depending on the age and gender of the population, the season, the region of residence, and the type of concomitant pathology. The purpose of the study was to assess the vitamin D status in the Ukraine population during 2016-2022 years depending on age, sex, month, and year of observation. In a single-center cohort study, serum 25-hydroxyvitamin D (25(OH)D) level was analyzed in 7105 subjects aged 20-99 years. The analysis was performed depending on age, sex, month, and year of observation. The mean serum 25(OH)D level in the total group was 30.9 [22.1-41.0] ng/ml, the lowest level was in the age group 90-99 years old and the highest one was in the subjects aged 40-69 years old. 52.7% of the subjects had a sufficient vitamin D level, 27.4% had insufficiency, and 19.9% had a deficiency of vitamin D. No gender differences were found in the serum level of 25(OH)D, except the one for the women aged 60-69 years old, who had higher vitamin D levels compared to males parameters. Seasonal 25(OH)D levels variations indicated the highest values in September and October and the lowest ones in February and March. Additionally, we established the increase of serum 25(OH)D from 2016 to 2021 with the highest values in 2020 and 2021. Our data confirmed a decrease in vitamin D deficiency and insufficiency in 2021 and 2022 in the Ukrainian population compared to previous years (2016-2019) and previous studies in the Ukrainian population while maintaining their age-related and seasonal characteristics. It may be associated with an improvement in public awareness of global vitamin D deficiency and its positive skeletal and extraskeletal effects, as well as the COVID-19 pandemic in recent years.