Searching for potential relations between changes in 25(OH)D concentration and in physical fitness is an interesting scientific topic. Thus, the main purpose of this study was to investigate the changes in serum concentrations of 25(OH)D in young football players in conjunction with indicators determining bone resorption and physical fitness. A total of 35 young soccer players were tested during the autumn competition period. Biochemical analysis of blood, aerobic capacity, running speed and power tests (Wingate test, squat jump, counter movement jump) were carried out at the beginning and at the end of the competition period. Significant decrements in concentration of 25(OH)D from 33.9 ± 5.87 to 23.7 ± 5.58 ng/mL were noted after the analyzed competition period. There were no significant changes in aerobic capacity along the competition period. Significant improvements were observed in 10 m sprint, 30 m sprint (p < 0.001), maximal power (p = 0.011) and total work capacity (p = 0.024). We found that the effect of changes in the players’ physical fitness does not occur in relation to 25 OH(D) concentration but occurs when these changes are analyzed as a function of the duration of the observation period. Changes in physical fitness of soccer players are determined by other factors then 25(OH)D concentration.