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Background: The worldwide increasing vitamin D deficiency in adolescents is alarming. The effect of high-intensity trainings on adolescent health is not clear. Object: This study aimed to examine the effect of a high-intensity interval training (HIIT) on plasma level of vitamin D in overweight male adolescents. Materials and Methods: About 96 overweight students aged 12–16 years from two high schools participated in the study. The schools were randomly assigned to the intervention group (n = 52) and the control group (n = 44). For the intervention group, HIIT was performed 3 days per week for 8 weeks, while the control group was asked to walk outdoors at the same time. Results: At the end of the study, the vitamin D level was increased to 1.21 ng/dl in the intervention group, whereas it decreased to 1.94 ng/dl in the control group (P = 0.003). Conclusion: About 8 weeks of HIIT improved plasma level of vitamin D in overweight male adolescents. More prolonged interventions consisted of both the sex and broader age ranges are warranted.