Skeletal muscle is a direct target for Vitamin D. Observational studies suggest that 25[OH]D correlates with recovery of skeletal muscle following eccentric contractions in humans and crush injury in rats. However, a definitive association is yet to be established. In order to address this gap in knowledge in relation to damage repair, a randomised, placebo-controlled trial was performed in twenty males with low serum 25[OH]D (45 ± 25 nmol.L^-1). Prior to and following 6-weeks of supplemental Vitamin D₃ (4,000 IU.day^-1) or placebo (50 mg cellulose), participants performed 20x10 damaging eccentric contractions of the knee extensors with peak torque measured over the following 7 days of recovery. Parallel experimentation using isolated human skeletal muscle derived myoblast cells from biopsies of 14 males with insufficient serum 25[OH]D (37 ± 11 nmol.L^-1) were subjected to mechanical wound injury, which enabled corresponding in vitro studies of muscle repair, regeneration and hypertrophy in the presence and absence of 10 nmol or 100 nmol 1α,25[OH]₂D₃. Supplemental Vitamin D₃ increased serum 25[OH]D and improved recovery of peak torque at 48 hours and 7 days post-exercise. In vitro, 10 nmol 1α,25[OH]₂D₃ improved muscle cell migration dynamics and resulted in improved myotube fusion/differentiation at the biochemical, morphological and molecular level together with increased myotube hypertrophy at 7 and 10 days post-damage. Together, these preliminary data are the first to characterise a role for Vitamin D in human skeletal muscle regeneration and suggest that maintaining serum 25[OH]D may be beneficial for enhancing reparative processes and potentially for facilitating subsequent hypertrophy.