BACKGROUND: Prosthetic patches can be used to repair large congenital diaphragmatic hernia defects but may be associated with infection, recurrence, and thoracic deformity. Biosheets (collagenous connective tissue membranes) have been used in regenerative medicine. We evaluated the efficacy of Biosheets in a rabbit model. METHODS: Biosheets were prepared by embedding silicone plates in dorsal subcutaneous pouches of rabbits for 4weeks. In group 1 (n=11), Gore-Tex® sheets (1.8×1.8cm) were implanted into a diaphragmatic defect. In group 2 (n=11), Seamdura®, a bioabsorbable artificial dural substitute, was implanted in the same manner. In group 3 (n=14), biosheets were autologously transplanted into the diaphragmatic defects. All rabbits were euthanized 3months after transplantation to evaluate their graft status. RESULTS: Herniation of liver was observed in 5 rabbits (45%) in group 1, 8 (73%) in group 2, and 3 (21%) in group 3. A significant difference was noted between groups 2 and 3 (P=0.017). Biosheets had equivalent burst strength and modulus of elasticity as native diaphragm. Muscular tissue regeneration in transplanted biosheets in group 3 was confirmed histologically. CONCLUSION: Biosheets may be applied to diaphragmatic repair and replacement of diaphragmatic muscular tissue. LEVEL OF EVIDENCE: Level III.