Objective To investigate the effects of exposure to glucocorticoids with different dosage on skeleton of normal 3-month-old rats by dual energy X-ray absorptiometry (DXA), biomechanical testing and bone histopathology. Methods Thirty-one 3-month-old female clean level SD rats were randomly divided into 3 GC-treated groups, with tail intravenous injections of dexamethasone (Dex) at the dosage of 1, 2.5, 5 mg/kg twice per week for 8 weeks, respectively, and 1 normal control group treated with saline. At the end of experiment, bone mineral content (BMC) and bone mineral density (BMD) of the femur and the 3rd lumbar vertebrae in rats were measured by DXA. The 3-point bending test of the total femur and compression test of the 5th lumbar vertebrae were also conducted, respectively. Microstructure of the trabecula in proximal metaphysis of the tibia was observed by bone pathological section for quantitative analysis. Results Compared with control group, the body weight was significantly decreased in all Dex-treated groups, while no obvious decrease in vertebral BMC, BMD and maximum compressive loads was found. The total femoral BMC was also reduced significantly in all Dex-treated groups, while the total femoral BMD, proximal and distal femoral BMD were reduced only in Dex 1mg group. The fracture load, maximum load and elastic load for 3-point bending test were all evidently decreased in Dex 1mg group, while the Dex 2.5mg group and Dex 5mg group only showed a decline in elastic load. All Dex-treated groups showed an unevenly spatial distribution of the trabecula, which indicated a low bone metabolic state. Conclusions The exposure to GC for 8 weeks brings negative effects on skeleton of the 3-month old rats, which will cause more bone loss and worse mechanical properties in femur than in lumbar vertebrae. Higher dosage of Dex does not increase bone mass loss or change the mechanical properties. Both the decline in bone mechanics, especially elastic load, and unevenly density distribution of trabecula indicate that Dex will affect more bone quality other than bone mass. The side effects of GCs on skeleton in clinical application should be evaluated with various methods.