This randomized controlled trial aimed to test whether vitamin D (VD) supplementation affects measures of physical performance in VD-insufficient mildly trained children. Thirty-six recreationally soccer player boys were randomly assigned to single dose (200,000 IU) of VD3 (n=19) or placebo (n=17). Plasma 25-hydroxyvitamin D (25-OHD) was assessed and measures of physical performance (i.e. vertical and standing broad jumps, triple hop, 10-m and 30-m sprints, shuttle run) were performed before and 12 weeks after the loading dose. Mixed ANCOVA models were performed and effect size was estimated by partial eta squared (ηp2). Baseline 25-OHD and physical variables were equivalent in the two groups. Twelve weeks after VD loading, plasma 25-OHD increased and physical variables improved in VD group, only. There was a significant interaction effects for group by time for vertical jump (F=14.9, p=0.001, ηp2=0.394), triple hop jump (F=24.2, p<0.001, ηp2=0.513), 10-m (F=4.46, p=0.046, ηp2=0.162) and 30-m (F=6.56, p=0.017, ηp2=0.222) sprints, and shuttle run (F=13.4, p=0.001, ηp2=0.369). In conclusion, a single bolus of VD3 resulted in significant improvements in jumping ability, agility, and running speed in VD-insufficient mildly trained children. The findings suggest that correcting VD deficit might be beneficial for physical performance. • A mega dose of vitamin D3 improves jumping ability, agility, and running speed in VD-insufficient mildly trained children • Effect of vitamin D on measures of physical performance is noticeable three months after the loading dose.