The specific orientation of collagen and biological apatite (BAp) is an anisotropic feature of bone micro-organization; it is an important determinant of bone mechanical function and performance under anisotropic stress. However, it is poorly understood how this microstructure orientation is altered when the mechanical environment changes. We hypothesized that the preferential orientation of collagen/BAp would change in response to changes in mechanical conditions, similar to the manner in which bone mass and bone shape change. In the present study, we investigated the effect of unloading (removal of anisotropic stress) on the preferential orientation of collagen/BAp using a rat sciatic neurectomy model. Bone tissue that formed under unloaded conditions showed a more disordered collagen/BAp orientation than bone tissue that formed under physiological conditions. Coincidentally, osteocytes in unloaded bone displayed spherical morphology and random alignment. To the best of our knowledge, this study is the first to demonstrate the degradation of preferential collagen/BAp orientation in response to unloading conditions. In summary, we identified alterations in bone material anisotropy as an important aspect of the bone's response to unloading, which had previously been examined with regard to bone loss only.