Bone tissue is a composite structure of apatite particles distributed in collagen fibril matrix on a nano-size scale. Mechanical properties of cortical bone are determined by the apatite-collagen composition. The amount and specific orientation of apatite crystals strongly affect the mechanical properties of macroscopic bone tissue such as anisotropic elastic modulus and fracture toughness. The progression of mineralization with tissue aging results in a reduction of fracture toughness due to embrittlement of tissue caused by excessive increase of apatite density. This study focused on the change of impact fracture characteristics of cortical bone tissue with reduction of apatite concentration (demineralization). A compact-sized impact loading device for Charpy tests was developed to measure the absorbed energy for impact fracture of bone specimens in the 0.5 and 1.0 J input energy range. Cortical bone specimens of 3 × 3 × 30 mm were prepared from shaft of bovine femurs. Differences relating to the anisotropy in axial and circumferential direction of the femur were observed in the absorbed energy values. The values of the axial specimens were greater than the circumferential specimens. Axial bone specimens were demineralized in ethylenediaminetetraacetic acid (EDTA) solution at 5°C. The demineralization progressed slowly from surfaces of the specimen. The 24-hour demineralization created a collagen layer at the surface of specimen and the demineralized specimen showed higher absorbed energy than unprocessed specimens. The absorbed energy in defective specimens with a square shaped small slit of 0.5 × 0.5 mm increased after local demineralization process. The time for effective demineralization could be reduced to 2 hours in the case of 37°C condition. The demineralization process improved the fracture characteristics of both intact and defective cortical bone tissue.