Research objective: The aim of this research was to examine whether there are differences in the concentration of 25(OH) vitamin D3 in subjects aged 18 to 28 by sex during winter and to compare the results obtained in this research with reference values and data from the literature. Subjects and methods: The research was conducted on a sample of 59 subjects, of which 27 (45.8%) were male and 32 (54.2%) were female. The subjects’ blood was extracted and centrifuged at 3,000 rpm for 10 minutes. After centrifugation, the serum was separated and the samples were analyzed by liquid chromatography–mass spectrometry (LC-MS/MS) using an LCMS-8050 mass spectrometer. Results: Men were found to have statistically significantly higher concentrations of 25-OH vitamin D3 than women. Average concentrations of vitamin D in the serum were 18.96 ng/mL for men and 14.16 ng/mL for women. Significant deviation from the 25-OH vitamin D reference values was detected in the group of women and in the entire sample of subjects (Student's t-test, P=0,01). The percentage of subjects who reached the target value (> 20 ng/mL) was 28,8 %, of which 44,4 % men and 15,6 % women. Conclusion: Men had statistically higher levels of 25(OH) vitamin D3 when compared with women. Significant deviation from the 25-OH vitamin D reference values was detected in the group of women and in the entire sample of subjects. A deficit of 25(OH) vitamin D3 during winter was observed in the subjects, which is in accordance with the data on vitamin D that can be found in the literature.