Objective: The purpose of this study was to investigate the effect of fresh human amniotic membrane (FHAM) as a substitute of periosteum to enhance bone regeneration in critical-sized defects.Methods: Tibial diaphyseal bone defects were created in forty New Zealand white rabbits and treated with FHAM or left empty. Treatment groups consisted of: FHAM implanted in the place of removed periosteum (FHAMP group); FHFAM implanted to fill the entire defect (FHAMF group) compared to negative control group; empty defect with removing the periosteum (NC group) and positive control group; and empty defect without removing the periosteum (PC group). Bone regeneration was evaluated by radiographic, micro-computed tomography (μ-CT) and histological analyses at 4 and 8weeks post-surgery.Results: Radiographic and μ-CT analysis demonstrated clearly enhanced new bone formation in positive control group (PC) and FHAMP group compared to negative control group (NC) and FHAMF group. Histological staining exhibited remaining woven bones and cartilage matrix in the FHAMP group, immature lamellar bone with medellury cavity and marrow bone formation in PC group from 4weeks post-operatively. For FHAMF group, a little new bone formation was detected only from 8weeks post-operatively, and an absence of any sign of healing in NC group at both time points.Conclusion: The results provide that FHAM increases bone regeneration in critical-sized defects when it is implanted in the place of the removed periosteum, but its additive effect does not have the same effect of the natural periosteum.Read less <