Vitamin D is necessary for musculoskeletal health, however, the supplementation of vitamin D above the sufficiency level does not bring additional bone mass density (BMD), unlike physical exercise which enhances the bone formatting process. Regular physical activity has been shown to upregulate VDR expression in muscles and to increase circulating vitamin D. Here we investigate whether a single bout of exercise might change 25(OH)D3 blood concentration and how it affects metabolic response to exercise. Twenty-six boys, 13.8 years old (SD ± 0.7) soccer players, participated in the study. The participants performed one of two types of exercise: the first group performed the VO2max test until exhaustion, and the second performed three times the repeated 30 s Wingate Anaerobic Test (WAnT). Blood was collected before, 15 min and one hour after the exercise. The concentration of 25(OH)D3, parathyroid hormone (PTH), interleukin-6 (IL-6), lactate, non-esterified fatty acids (NEFA) and glycerol were determined. 25(OH)D3 concentration significantly increased after the exercise in all boys. The most prominent changes in 25(OH)D3, observed after WAnT, were associated with the rise of PTH. The dimensions of response to the exercises observed through the changes in the concentration of 25(OH)D3, PTH, NEFA and glycerol were associated with the significant increases of IL-6 level. A single bout of exercise may increase the serum’s 25(OH)D3 concentration in young trained boys. The intensive interval exercise brings a more potent stimulus to vitamin D fluctuations in young organisms. Our results support the hypothesis that muscles may both store and release 25(OH)D3.