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 randomised controlled trials with adequate statistical power to test this relationship, and none in the southern hemisphere. Thus, a prospective double blind, randomised placebo-controlled intervention trial was conducted, involving 57 professional rugby union players in New Zealand. Participants were randomised 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 to 38; P < 0.001). Performance in five of the six tests, including the primary outcome variable of 30m 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 to 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.