Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopedic surgeons and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. At the same time, a variety of disadvantages still remain. The present study has been designed in vivo to evaluate the effects of osteogenic medium on healing of experimental critical bone defect in a rabbit model. Twenty New Zealand albino rabbits, 12 months old, of both sexes, weighing 2.0±0.5 kg were used in this study. An approximately 10mm segmental defect was created in the mid portion of each radius as a critical size bone defect. In the osteogenic medium group (n=5) 1 ml osteogenic medium, in the maintenance medium group (n=5) 1 ml maintenance medium, and in the normal saline group (n=5) 1 ml normal saline were injected in the defected area while the defects of the rabbits of the control group (n=5) were left empty. Radiological evaluation was done on the 1st day and then at the 2nd, 4th, 6th and 8th weeks post injury. Biomechanical and histopathological evaluations were done 8 weeks post injury. The radiological, histological and biomechanical findings of the present study indicated a superior bone healing capability in the osteogenic and maintenance medium groups, by the end of 8 weeks post-surgery, in comparison to the normal saline and control groups. In conclusion, this study demonstrated that the osteogenic medium and maintenance medium could promote bone regeneration in long bone defects better than the control group in rabbit model.