Vitamin D insufficiency is common in athletes and may lower physical performance. Many cross-sectional studies associate vitamin D status with physical performance in athletes; however, there have been few prospective randomized controlled trials with adequate statistical power to test this relationship, and none in the southern hemisphere. Thus, a prospective double-blind, randomized, placebo-controlled intervention trial was conducted involving 57 professional rugby union players in New Zealand. Participants were randomized to receive 50,000 IU of cholecalciferol (equivalent to 3,570 IU/day) or placebo once every two weeks over 11–12 weeks. Serum 25(OH)D concentrations and physical performance were measured at baseline, weeks 5–6, and weeks 11–12. Mean (SD) serum 25(OH)D concentrations for all participants at baseline was 94 (18) nmol/L, with all players above 50 nmol/L. Vitamin D supplementation significantly increased serum 25(OH)D concentrations compared to placebo, with a 32 nmol/L difference between groups at 11–12 weeks (95% CI, 26–38; p < 0.001). Performance in five of the six tests at study completion, including the primary outcome variable of 30-m sprint time, did not differ between the vitamin D supplemented and placebo groups (p > 0.05). Performance on the weighted reverse-grip chin up was significantly higher in players receiving vitamin D compared with placebo, by 5.5 kg (95% CI, 2.0–8.9; p = 0.002). Despite significantly improving vitamin D status in these professional rugby union players, vitamin D supplementation had little impact on physical performance outcomes. Thus, it is unlikely that vitamin D supplementation is an ergogenic aid in this group of athletes.