Aim: This study aimed to evaluate serum levels of 25-OH D3 (calcidiol) among students of the Faculty of Medicine Osijek, Croatia, thereby determining to what extent vitamin D deficiency is present. Methods: The present cross-sectional analysis was based on data collected from 60 participants. Blood sampling was done in March 2021. Concentrations of 25-OH D3 were measured using LC/MSMS procedure on Shimadzu LCMS 8050 and RECIPE kit for serum levels of 25-OH-D3. Results: The study was conducted on a sample of 60 respondents aged 19 to 28, of whom 16 were men and 44 were women. All subjects had a 25-OH D3 deficiency (<20 ng/ml), while 11 had an extreme deficiency. Mean 25-OH D3 concentration was 11.36 ng/ml, ranging from 7.70 ng/ml to 16.70 ng/ml. There was no statistical significance of 25-OH D3 concentration levels between the sexes (Student’s t-test, P>0.05). Conclusion: Vitamin D deficiency was detected in all subjects. In addition to the results of several other studies conducted worldwide that evaluated vitamin D status among medical students, this study further highlights the problem affecting this student subgroup.