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Background: Silk cocoon is composed of multiple layers. The natural silk cocoon containing all layers was cut asa rectangular shape as defined as total group. The inner and outermost layers were removed from the total groupand the remained mat was defined as the middle group. The objectives of this study was to compare the totalgroup with the middle group as a barrier membrane for the guided bone regeneration. Methods: The effects of these materials on the cellular proliferation and alkaline phosphatase (ALP) expression ofMG63 cells were explored. For comparing bone regeneration ability, bilateral bone defects were created in calvarialareas in ten adult New Zealand white rabbits. The defects were covered with silk membranes of the middle group,with silk membrane of the total group used as the control on the contralateral side. The defects were allowed toheal for 4 and 8 weeks. Micro-computerized tomography (μCT) and histological examination were performed. Results: The middle group exhibited a higher MTT value 48 and 72 h after treatment compared to the total group. ALPexpression was also higher in the middle group. The results of μCT and histologic examination showed that new boneformation was significantly higher in the middle group compared to the total group 8 weeks postoperatively (P < 0.05). Conclusions: In conclusion, the middle layer of the silk cocoon supports guided bone regeneration better thanunprocessed silk cocoon.