In this study, we evaluated the effect of functional disuse-induced bone remodeling on its mechanical properties, individually at periosteum and medullary endosteum regions of the cortical bone. Left middle tibiae were obtained from 5-month-old female Sprague–Dawley rats for the baseline control as well as hindlimb suspended (disuse) groups. Micro-nano-mechanical elastic moduli (at lateral region) was evaluated along axial (Z), circumferential (C) and radial (R) orientations using nanoindentation. Results indicated an anisotropic microstructure with axial orientation having the highest and radial orientation with the lowest moduli at periosteum and medullary endosteum for both baseline control as well as disuse groups. Between the groups:​ at periosteum, an insignificant difference was evaluated for each of the orientations (p > 0.05) and at endosteum, a significant decrease of elastic moduli in the radial (p < 0.0001), circumferential (p < 0.001) and statistically insignificant difference in axial (p > 0.05) orientation. For the moduli ratios between groups:​ at periosteum, only significant difference in the Z/R (p < 0.05) anisotropy ratio, whereas at endosteum, a statistically significant difference in Z/C (p < 0.001), and Z/R (p < 0.001), as well as C/R (p < 0.05) anisotropy ratios, was evaluated. The results suggested initial bone remodeling impaired bone micro-architecture predominantly at the medullary endosteum with possible alterations in the geometric orientations of collagen and mineral phases inside the bone. The findings could be significant for studying the mechanotransduction pathways involved in maintaining the bone micro-architecture and possibly have high clinical significance for drug use against impairment from functional disuse.