Stress fractures are common amongst healthy military recruits and athletes. Reduced vitamin D availability, measured by serum 25-hydroxyvitamin D (25OHD) status, has been associated with stress fracture risk during the 32-week Royal Marines (RM) training programme. A gene-environment interaction study was undertaken to explore this relationship to inform specific injury risk mitigation strategies. Fifty-one males who developed a stress fracture during RM training (n = 9 in weeks 1-15; n = 42 in weeks 16-32) and 141 uninjured controls were genotyped for the vitamin D receptor (VDR) FokI polymorphism. Serum 25OHD was measured at the start, middle and end (weeks 1, 15 and 32) of training. Serum 25OHD concentration increased in controls between weeks 1-15 (61.8±29.1 to 72.6±28.8 nmol/L, p = 0.01). Recruits who fractured did not show this rise and had lower week-15 25OHD concentration (p = 0.01). Higher week-15 25OHD concentration was associated with reduced stress fracture risk (adjusted OR 0.55[0.32-0.96] per 1SD increase, p = 0.04): the greater the increase in 25OHD, the greater the protective effect (p = 0.01). The f-allele was over-represented in fracture cases compared with controls (p<0.05). Baseline 25OHD status interacted with VDR genotype: a higher level was associated with reduced fracture risk in f-allele carriers (adjusted OR 0.39[0.17-0.91], p = 0.01). Improved 25OHD status between weeks 1-15 had a greater protective effect in FF genotype individuals (adjusted OR 0.31[0.12-0.81] vs. 1.78[0.90-3.49], p<0.01). Stress fracture risk in RM recruits is impacted by the interaction of VDR genotype with vitamin D status. This further supports the role of low serum vitamin D concentrations in causing stress fractures, and hence prophylactic vitamin D supplementation as an injury risk mitigation strategy.