Bone loss has been supposed to be the greatest damage to the health of astronauts. It is generally believed that the mechanical unloading induced by microgravity is the main cause of bone loss. However, besides mechanical unloading, many evidences from animal models and spaceflight missions indicate that microgravity conditions can cause some stress reactions and elevated endogenous glucocorticoid (GC) levels. High levels of GCs can lead to bone loss. This study aimed to investigate whether elevated GC levels are involved in hindlimb unloading (HLU)-induced bone loss in mice. Col2.3-11β-hydroxysteroid dehydrogenase type 2 (Col2.3-11β-HSD2) transgenic mice which are characterized by specific blocking GC signaling in mature osteoblasts and osteocytes were used. Male 14-week-old Col2.3-11β-HSD2 transgenic mice and wild type littermates were tail-suspended or kept under ambulatory conditions. At the endpoint, the tibias were examined by micro-computed tomography and histomorphometry, and bone turnover was analyzed by serum biochemistry, histochemistry staining, immunohistochemistry, and real-time PCR. Mice exposed to unloading occurred a significant increase in serum GC concentrations. Compared with non-unloaded controls, HLU led to a severe damage in cortical bone microstructure and bone strength of the tibia in wild type mice but not transgenic littermates. Osteoblast activity and bone formation were inhibited, whereas osteoclast activity and bone resorption were promoted in the tibial cortical bone of wild type mice following HLU, features absented in transgenic mice. Furthermore, HLU resulted in a significant increase in the number of sclerostin-producing and receptor activator of nuclear factor-κ B ligand (RANKL)-positive osteocytes, and apoptotic osteoblasts and osteocytes in wild type mice of unloading but not in unloaded transgenic mice. In conclusion, cortical bone loss during HLU is mediated through enhancing GC signaling in osteoblasts and osteocytes and subsequently restraining bone formation and activating bone resorption. It suggests that elevated GC levels play an important role in cortical bone loss in response to mechanical unloading.