To evaluate the material parameters of regenerated bone, it is important to clarify the mechanical performance of the regenerated portion. In general, the shape and size of regenerated bone tissue is heterogeneous. It is often difficult to elucidate material properties by means of conventional mechanical tests such as compressive and/or tensile tests and bending tests. The nanoindentation technique has been utilized to evaluate the material properties of small or microstructured materials because they do not necessarily require a large well-designed specimen. Thus, this technique may be useful for the evaluation of the material properties of regenerated bone tissue. In this study, this technique was applied for the assessment of the Young’s modulus and hardness of regenerated and intact long bones of a rabbit. The regenerated bone exhibited a significantly lower Young’s modulus and hardness than the intact bone. The regenerated long bone also exhibited impaired mechanical properties, which may have been caused by the difference in the nano-organization of its collagen fibers and mineral crystals (the main components of bone tissue), from that of the intact bone.