This study was aimed at elucidating the temporal changes of microarchitectural and mechanical parameters of cancellous bone in the osteoporotic rabbit model induced by ovariectomy (OVX) combined with glucocorticoid (GC) administration. Osteoporotic (OP) group received bilateral OVX combined with injections of GC, while sham group only received sham operation. Cancellous bone quality in vertebrae and femoral condyles in each group was assessed by DXA,μCT, nanoindentation, and biomechanical tests at pre-OVX and 4, 6, and 8 weeks after injection. With regard to femoral condyles, nanoindentation test could detect significant decline in tissue modulus and hardness at 4 weeks. However, BMD and microarchitecture of femoral condylar cancellous bone changed significantly at 6 weeks. In vertebrae, BMD, microarchitecture, nanoindentation, and biomechanical tests changed significantly at 4 weeks. Our data demonstrated that temporal changes of microarchitectural and mechanical parameters of cancellous bone in the osteoporotic rabbit were significant. The temporal changes of cancellous bone in different anatomical sites might be different. The nanoindentation method could detect the changes of bone quality at an earlier stage at both femoral condyle and vertebra in the osteoporotic rabbit model than other methods (μCT, BMD).