Objective Three-dimensional ( 3D) clinostat has been widely used to simulate the effect of microgravity effect on plants, cells, and nematodes. Although its mechanism in models other than rodents has been discussed, no rodent model has been established and discussed mechanism. Methods Thirty 8-week-old male C57BL/ J mice were randomly divided into three groups: independent ventilation cage group (MC, n= 6), survival box group (SB, n= 12), and 3D clinostat group (CS, n= 12). After the study, loss of the left femur was detected using Micro-CT and serum metabolite changes were tested using metabolomics. Results After 3D clinostat treatment, the trabecular number decreased (P< 0. 05) and trabeculae spacing increased (P< 0. 5) in the CS group. Eighty-six metabolites in the CS group exhibited significant differences in metabolomics. These differential metabolites were mainly involved in protein digestion and absorption, and mineral absorption. Conclusions 3D clinostat microgravity induced bone loss and changes in metabolites and metabolic pathways in C57BL/ J mice.