Vitamin D is a derivative of cholesterol. Lack of this vitamin causes some disorders in bone metabolism. In this study, 32 adult female New Zealandian white rabbits were selected and divided into four equal groups randomly. After the induction of general anesthesia, a full thickness defect with 1 mm width was created in the mid shaft of ulnar bone of both right and left for limbs in all groups. The animals in first, second and third groups received the amount of 2500 IU, 5000 IU and 10000 IU vitamin D3/kgBW via intramuscular injection, respectively. Then, the same dose was repeated for them once in a week during 50 days. The animals in the fourth group received equal volume of normal saline as same as above regimes. 50 days after surgery, the rabbits become Euthanasia. Then bone defect was evaluated by means of histopathological, and biomechanical factors. Biomechanical evaluation showed that the mean force required breaking bones in group 2 was higher and it was equivalent to 124.75±2.13 N. Histopathological evaluation showed that bone repair in group 2 was performed better than other groups. Considering the results of this study, the 1, 25-dihydroxyvitamin D3 has an effective role on repairing bone defect; but in high dose, it can prevent mineralization of bone.