Objective To explore biomechanical assessment for the effects on vibration against bone loss by investigating the relationship between material distribution and mechanical properties of rat femur cortical bone based on Micro CT. Methods 35 rats were randomly divided into intermittent vibration groups with the interval of 1, 3, 5, 7 days, and continuous vibration group, respectively. Tail suspended model of disuse osteoporosis was set up. All rats were loaded with mechanical vibration of 35 Hz and 0.3 g, and killed after 8 weeks. Micro CT scanning of the left femur of each rat was performed. Three-dimensional finite element model of the cortical bone was established to calculate the apparent and tissue-level mechanical parameters. Principal components (PCs) were extracted from material distribution, intermittent days and volume fraction by principal components analysis (PCA). Results The PCA revealed the three independent components that could fully explaine the variability of cortical bone characteristics under vibration. The linear regression equations were also created between the material property and the apparent and tissue-level mechanical properties, respectively. Mechanical properties of the cortical bone were influenced by material distribution mostly, and the volume fraction and intermittent days were next in importance. Conclusions The cortical bone material distribution can reflect changes in its mechanical properties, and the bone strength could be assessed by establishing the linear relationship, which could provide a theoretical basis for osteoporosis prevention and treatment as well as the assessment on its rehabilitation process.