Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix on bone regeneration in a mouse bone defect model of the calvaria. Matrigel was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix-injected bone defects was stronger than that in the Matrigel-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix, but not in the Matrigel-treated defects. Notably, PuraMatrix treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix than Matrigel. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix (146.0 g) than for Matrigel (84.7 g). The present study demonstrated that PuraMatrix injection favorably induced functional bone regeneration.