Mechanical properties of bone are essential to understand the changes in bone fracture risk with aging and/or osteoporosis and to evaluate the effects of osteoporotic medicines as well as bone volume fraction and bone mineral density. The relationship between mechanical properties and hierarchical structure of cortical bone has been investigated using the bone samples taken from an inner region of the diaphysis. According to an optical microscopic observation, different microstructure was observed between cortical surface and inner region of the diaphysis. Hence, the study aimed to investigate the distributions of elastic modulus, microstructure, hydroxyapatite (HAp) crystal orientation, and mineral contents from the cortical surface to the inner region of the bovine femoral diaphysis. In the experiments, strip bone specimens were taken from the surface and the inner region of bovine femoral diaphyses. As a result, elastic modulus at the surface region was significantly lower than that in the inner region (73%). Osteon area fraction at the surface region was significantly lager than that in the inner region (141%). On the other hand, HAp crystal orientation showed no significant difference. Mineral contents at the surface were slightly lower than that in the inner region (98%). It suggests that elastic modulus of osteon region is lower than that of non-osteon region and that the difference of elastic modulus between surface and inner region may depend on the osteon area fraction as well as mineral contents.