The favorable biological properties of silk fibroin (SF) nanofiber membrane make it a good candidate for clinical applications as a device in bone and periodontal regenerative therapy. The purpose of this study is to evaluate the biocompatibility of the SF nanofiber membrane, and to examine its effect on bone regeneration in a rabbit calvarial model. To examine the biocompatibility of the electrospun SF membrane, we investigated cell proliferation, morphology, and differentiation. The bone regenerative efficacy of the membrane was evaluated in the calvarial defect of rabbits. The cell numbers and osteocalcin production labels were significantly increased in accordance with culture period. Cells had a stellate shape and broad cytoplasmic extensions on the membrane. The cells showed activity of ALPase that was comparable to culture dishes, and were calcified similarly to culture dishes. In in vivo tests, a complete bony union across the defects was observed after 8 weeks. At 12 weeks, the defect had completely healed with new bone. In conclusion, the SF nanofiber membrane was shown to possess good biocompatibility with enhanced bone regeneration and no evidence of any inflammatory reaction. These results strongly suggest that the SF membrane should be useful as a tool for guided bone regeneration.