©2019 The Japan Institute of Metals and Materials In this study, we focused on the preferential orientation of the extracellular matrix (ECM) of bone, since ECM orientation has been shown to significantly affect the mechanical functions of bones. Bone analysis is in most cases based on the premise that the apatite crystallizes on the collagen template such that its c-axis is parallel with the running direction of the collagen fibril. Bone regeneration analysis has also been discussed assuming that the apatite c-axis orientation reflects collagen orientation. To understand the regeneration processes of both collagen and apatite individually, the preferential orientations of apatite and collagen in regenerated bone were simultaneously analyzed using a bone regeneration model of mouse femur with an 0.8-mm drill hole defect. The defects in mouse femur were filled with mineralized bone matrix, which shows an intact mineral density. However, the directions of orientation of the collagen and apatite deviate from the femur longitudinal axis in the regenerated bone. Moreover, electron diffraction analysis revealed that the apatite c-axis aligned along the extended axis of a collagen fibril both in regenerated and intact bones, indicating that the direction of the apatite c-axis is regulated by collagen fibril orientation even in the regenerated bone. In conclusion, the less-oriented apatite crystallite observed in the regenerated bone was shown to be formed due to the less-oriented collagen fibrils.