Objective: Bone has complex hierarchical structure. Investigating the changes in microstructure of osteocyte lacuna-canalicular network at cell scale in osteoporotic rats is of significance to understand the imbalance of bone remodeling in osteoporosis, and to explore the causes of bone loss and bone mineral density decline of cortical bone and cancellous bone at tissue scale in osteoporotic rats. The aim of this study was to analyze the changes in bone microstructure of osteoporotic rats at tissue and cell scales. Methods: Twenty 5-month-old female SD rats were randomly divided into two groups, i.e., ovariectomy (OVX) group (n=12) and the SHAM group (n=8), respectively. The rats in OVX group were subjected to bilateral ovariectomy, while sham operation was performed for the SHAM group. Changes in microstructure of cortical bone and cancellous bone at tissue scale, and osteocyte lacuna-canalicular network and extracellular matrix at cell scale were quantitatively analyzed using Micro-CT and SR-Nano-CT after the postoperative 8 weeks. Results: At tissue scale, the cross-sectional area of cortical bone in OVX group was significantly higher than that in SHAM group (p<0.05), and the bone mineral density (BMD) and thickness of cortical bone were not significantly different from those in SHAM group. The trabecular BMD, bone volume fraction, trabecular thickness and trabecular number in OVX group were significantly decreased in comparison with SHAM group (p<0.01), while the trabecular separation was significantly increased (p<0.01). At cell scale, there was no significant difference in the semiaxes of lacunae between OVX group and SHAM group, but the thickness of lacunae and the diameter of canaliculi in OVX group were significantly increased in comparison with SHAM group (p<0.05). At the same time, the porosity of cortical bone in OVX group was significantly higher than that in SHAM group at the cell scale (p<0.05). Conclusion: Microstructure of bone in OVX group varied to different extents at tissue and cell scales. At tissue scale, the cancellous bone loss was severe, while the cortical bone had little changes. At cell scale, porosity of the lacunar-canalicular network significantly increased, which directly affected the BMD and strength of cortical bone. Multiscale analysis of the changes in bone microstructure of osteoporotic rats has potential application value for clinical diagnosis and pathological analysis of osteoporosis.