<p>Disuse conditions, such as microgravity, prolonged bed rest, sedentary lifestyle, and limb immobilization, frequently affect human skeletal responses to mechanical loading. However, the precise impact of disuse on bone adaptation to mechanical loading and its underlying mechanobiological mechanisms remain unclear. Osteocytes are recognized as the primary mechanosensors within the fluid dynamic microenvironment of the lacunocanalicular network (LCN). Given that disuse can significantly alter LCN structure, we hypothesized that disuse would influence cancellous and cortical bone responses to mechanical loading by modifying lacunocanalicular fluid dynamics. To test this hypothesis, we employed a combination of in vivo animal experiments, whole bone-LCN multi-scale modeling, and RNA-sequencing. By applying identical in vivo axial loading to tibiae of hindlimb unloading and ground control mice, we observed that disuse enhanced loading-induced changes in cancellous and cortical bone mass and mechanical properties. Furthermore, we discovered that disuse increased lacunar volumes in both cancellous and cortical bone, thereby enhancing the loading-induced fluid dynamic microenvironment (e.g., fluid shear stress [FSS]) for osteocytes and promoting activation of downstream FSS-related signaling pathways. These findings underscore the importance of the mechanical microenvironment of osteocytes within the LCN in regulating bone adaptive responses to mechanical loading. This study advances our understanding of bone mechanoadaptation, particularly under disuse conditions.</p>