In the USA, as many as 20 % of recruits sustain stress fractures during basic training. In addition, approximately one-third of female recruits develop Fe deficiency upon completion of training. Fe is a cofactor in bone collagen formation and vitamin D activation, thus we hypothesised Fe deficiency may be contributing to altered bone microarchitecture and mechanics during 12-weeks of increased mechanical loading. Three-week old female Sprague Dawley rats were assigned to one of four groups: Fe-adequate sedentary, Fe-deficient sedentary, Fe-adequate exercise and Fe-deficient exercise. Exercise consisted of high-intensity treadmill running (54 min 3×/week). After 12-weeks, serum bone turnover markers, femoral geometry and microarchitecture, mechanical properties and fracture toughness and tibiae mineral composition and morphometry were measured. Fe deficiency increased the bone resorption markers C-terminal telopeptide type I collagen and tartate-resistant acid phosphatase 5b (TRAcP 5b). In exercised rats, Fe deficiency further increased bone TRAcP 5b, while in Fe-adequate rats, exercise increased the bone formation marker procollagen type I N-terminal propeptide. In the femur, exercise increased cortical thickness and maximum load. In the tibia, Fe deficiency increased the rate of bone formation, mineral apposition and Zn content. These data show that the femur and tibia structure and mechanical properties are not negatively impacted by Fe deficiency despite a decrease in tibiae Fe content and increase in serum bone resorption markers during 12-weeks of high-intensity running in young growing female rats.