Vitamin D (vit D) deficiency is common in the elderly, and the aim of this study was to investigate whether vit D deprivation in ovariectomized (ovx) and normal rats would reduce fracture strength. Forty mature female Wistar rats were randomized into four groups: two were ovariectomized (ovx) and two were sham-operated (sham). One ovx and one sham group were fed a vit D-deficient diet (Ovx-D and Sham-D), and the control groups were fed normal rat chow (Ovx and Sham) for 12 weeks. Vit D deficiency was substantiated after 12 weeks by undetectable serum concentrations of 25OHD in the Sham-D and Ovx-D groups. 85Sr activity was lower in Sham-D than in the other groups (P < 0.005). Tibial and femoral weights and lengths showed no differences. Distal tibial trabecular bone volume was reduced in both ovx groups compare with sham (P < 0.005). Bone mineral density (BMD) was higher in sham than in Sham-D and both ovx groups (P < 0.005). Femoral area moment of inertia increased and ultimate stress decreased in Ovx-D compared with ovx (P < 0.05). Other biomechanical properties of the femoral shafts did not differ significantly. The femoral neck was significantly weaker in Ovx-D than in the other groups. In conclusion, ovx decreased tibial trabecular bone volume and both ovx and vit D depletion reduced femoral BMD in rats. Vit D depletion reduced the ultimate stress in the femoral shaft, and the combined depletion of estrogen and vit D significantly reduced the fracture strength in the femoral neck. This fits well with clinical evidence of how postmenopausal status combined with vit D deficiency lead to an increased risk of hip fractures, making this animal model a possible tool for investigating measures to prevent such fractures.