Osteopetrotic bone shows dissociation between bone mineral density (BMD) and bone strength. In this study, volumetric BMD; preferential orientation of the extracellular matrix (ECM), which is composed of collagen fibers and apatite crystals as bone material quality; and mechanical properties of the src osteopetrotic and normal mouse femoral cortical bone were analyzed and compared with each other at a bone tissue level. The degree of preferential orientation of ECM along the femoral long axis was significantly decreased in the src mice femur, suggesting deteriorated bone quality. Young's modulus, as a tissue-level mechanical property analyzed by nano-indentation technique along the long bone direction, also was decreased in the src mice cortical femur, in spite of the similar volumetric cortical BMD. To the best of our knowledge, this is the first report to demonstrate the synchronous deterioration of Young's modulus and anisotropic ECM organization in the src osteopetrotic mouse bone. These results indicate that the deterioration of the preferential ECM orientation is one major cause of the impaired mechanical property in the src mouse bone.