A study was conducted to evaluate the three-dimensional clinostat simulated microgravity effect on mouse models, focusing on the central nervous system. Eighteen mice were divided into three groups: control, survival box, and clinostat + survival box. Behavioral tests, femur micro-CT, brain transcriptomics, serum metabolomics, and fecal microbiomics were performed. Results showed decreased activity, altered gait, enhanced fear memory, bone loss, immune/endocrine changes in brain transcriptome, and altered metabolic pathways in serum and gut microbiota in clinostat-treated mice. The model closely mimics spaceflight-induced transcriptome changes, suggesting its value in studying microgravity-related neurological alterations and highlighting the need for attention to emotional changes in space.