Purpose. The aim of our study was to assess the relationship between 25(OH)D levels and skinfold thickness in Poland’s premier league (Ekstraklasa) football players. Methods. We enrolled 43 Poland’s premier league football players. The mean age was 22.7 ± 5.3 years. Serum levels of 25(OH)D were measured by electrochemiluminescence (ECLIA) using the Elecsys system (Roche, Switzerland). Skinfold measurements were taken with a Harpenden-type skinfold calliper body fat tester, characterised by a constant pressure of 10 g/mm². Results. Our study showed decreased serum 25(OH)D levels in 74.4% of the professional football players. We did show a statistically significant negative correlation between the 25(OH)D levels and the biceps skinfold thickness (r = −0.33), iliac crest skinfold thickness (r = −0.43), the sum of all the studied parameters (r = −0.5) and percentage of body fat (r = −0.49). Conclusions. Our results suggest that athletes with higher content of the adipose tissue may be at a higher risk of vitamin D deficiency, and that 25(OH)D levels need to be monitored in professional athletes, particularly during the winter season.