Measurements of bone mineral density (BMD) cannot accurately predict the risk of bone fracture in some clinical cases; however, BMD is a useful index for assessing the bone condition. Recently, various parameters related to bone strength have been investigated. Among them, we have focused on the preferential orientation of biological apatite (BAp) crystallites analyzed by microbeam-X-ray diffraction, a powerful tool for analyzing BAp crystallites in bones. BAp, a dominant component of bone, is an anisotropic ionic crystal with a hexagonal lattice. In this study, we investigated the mechanism underlying BAp orientation during endochondral or membranous ossification by administering macrophage colony-stimulating factor (M-CSF) to osteopetrotic (op/op) mice lacking M-CSF. op/op mice were treated with intraperitoneal injections of 5 μg recombinant human M-CSF (rhM-CSF); the first injection was administered on the 14th day after birth. In the treated op/op mice, the bone marrow cavities expanded significantly, and this expansion was accompanied by an increase in the number of osteoclasts. Moreover, the degree of BAp orientation along the longitudinal axis was higher in the treated group than in the untreated group. These results suggest that M-CSF is one of the important parameters controlling the preferential alignment of the BAp c-axis.