Bone graft had been widely investigated for reconstruction of bone defects or acceleration of bone healing in orthopedics, neurosurgery and dental surgery. Autograft is the golden standard of bone graft but it is associated with donor site morbidity and is restricted in quantity. Xenograft has been researched an alternative method for autograft. The purpose of this study was to investigate the efficacy of new bone formation according to three different preparations of implants on rabbit xenograft. Cortical bone xenografts which made from bovine femoral cortical bone were treated by freezing, freeze-drying or defat-freezing implant preparations. They were transplanted into proximal diaphyseal shaft of bifibulae of 15 rabbits which were divided into three groups according to their implant preparation method. The fibulae transplantations were evaluated radiographically and examined osteoblast activity by bone alkaline phosphatase (BALP) biweekly for 16 weeks to observe new bone formation and union of the experimental defected region. New bone formation was observed in 7 cases in freeze-drying and defat-freezing group, respectively. Union of proximal and distal end of defected region, which was considered as success of bone graft, was observed in 4 cases (40%; 4 of 10 cases), respectively. In freezing group, new bone formation was observed in 6 cases but, there is no union observed. BALP value was increased over twice after two weeks of graft procedure in all union cases of freeze-drying and defat-freezing group (two of five animals, respectively) then gradually decreased to 16th week. In non-union cases, there is no significant variation in BALP value. Defat-freezing or freeze-drying preparations of implants are more efficacious in new bone formation than freezing method on rabbit xenograft. While it is difficult to propose which is superior between defat-freezing and freeze-drying, defatting of implants may enhance new bone formation in xenograft.