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Objective To explore the dynamic changes of femur microstructure in ovariectomized osteoporosis mice at different stages. Methods Female C57 mice (8 weeks old) were randomly divided into sham-operation (Sham) and ovariectomy (OVX) groups. Each group was subdivided into 1-week, 1-month and 3-month subgroups on the basis of different postoperative time, which representeing early, middle and late menopause, respectively. The femurs were extracted for HE staining, immunohistochemistry and micro-CT analysis, and the changes in the bone microstructure of cancellous bone in the distal femurs, cortical bone in the middle femurs and the levels of osteogenic associated proteins in each group were evaluated. Results Compared with the Sham group, the bone microstructure of the OVX group showed no significant destruction at 1 week after operation, trabecula fracture and bone marrow cavity enlargement from 1 month to 3 months after operation, and significant changes in CT bone parameters BV/TV, Tb.N and Tb.Sp (P < 0.01, P < 0.001) except Tb.Th (P > 0.05). In the Sham group, the bone mass was significantly reduced until 3 months after operation compared with that in the 1-week group (P < 0.01). Bone loss in the OVX groups was worsened in a time dependent manner (P < 0.05, P < 0.01, P < 0.001). The expression level of Runx2 by immunohistochemistry was consistent with the results of micro-CT analysis. No significant differences were found in morphologic indices of cortical bone between the Sham subgroups and the OVX subgroups (P > 0.05). Conclusion Trabecular bone microarchitecture is impaired progressively after OVX operation in 8-week-old C57 mice from 1 week to 1 month, prior to cortical bone. This period can be regarded as the key time for studies on pathogenesis and prevention and treatment of osteoporosis.