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Background: The aim of this paper was to evaluate the change in 25-hidroxyvitamin D (25(OH)D) levels before and during the COVID-19 pandemic., Methods: In this retrospective, cross-sectional and methodological study included 86,772 patients (18-75 years) samples who were admitted to the Izmir Dokuz Eylul University Hospital (latitude and longitude (Turkey): 27 E 09; 38 N 25, respectively) for various reasons and whose 25(OH)D levels were measured in the biochemistry unit between 2019-2020 and 2020-2021 (before and during the COVID-19 outbreak). A time series analysis of monthly averages for 25(OH)D was performed. For the purpose of seasonal study, the mean levels of 25(OH)D are grouped by years. Data were modeled in terms of 25(OH)D levels using the MATLAB Curve Fitting Toolbox., Results: There was no significant difference between the sexes according to 25(OH)D levels (p>0.05). 25(OH)D levels were significantly higher in the summer months and lower in the winter months (p<0.001). When comparing the spring months, 25(OH)D levels in 2020 (18 ± 10) were found to be significantly lower than in 2019 (22 ± 12) (p<0.001); on the contrary, when examined based on the summer, autumn, and winter months, it was determined that 25(OH)D levels increased in 2020 (summer: 25 ± 13, autumn: 25 ± 14, and winter: 19 ± 10) compared to 2019 (summer: 23 ± 11, autumn: 22 ± 10, and winter: 19 ± 11) (p<0.001). In the estimates curve obtained with an error margin of 11% in the time series analysis, it was estimated that the 25(OH)D averages after the pandemic would be similar to those before the pandemic., Conclusions: Restrictions, partial or complete closures, and curfews can significantly affect individuals' 25(OH)D levels during the COVID-19 outbreak. There is a need for multicenter studies with larger populations covering different regions to strengthen and support our results., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Arayıcı et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)