Objective To investigate the role and influence of physiological loading and overloading on microgravity-induced osteoporosis, so as to find a reliable way to prevent or treat related-orthopedic disorders in astronauts induced by long-time space activity. Methods The microgravity environment in space was simulated by tail-suspension experiment, then the osteoporosis models of mice were built. A total of 32 C57BL/6J mice were randomly and evenly separated into four groups: normal group (normal), tail-suspension group (TS), physiological loading group (loading) and overloading group (overloading). Periodic dynamic mechanical load was applied on the left tibia in loading group and overloading group during tail-suspension test. After four weeks, tibial mechanical properties, micro-parameters of bone trabecular, biochemical indices and osteogenesis-related gene expression in each group were compared and analyzed. Results A great loss of tibial cancellous bone, significantly lower tibial biomechanical expression, serious damage of microstructure and weaker osteogenic activity were found in tail-suspended mice as compared with those of normal group. Physiological loading could clearly improve mechanical properties of bones, microstructure of bone trabecular, osteogenic activity and relative gene expression (P＜0.05). Overloading could also improve the condition of microgravity-induced osteoporosis, but the effect was not obvious (P＞0.05). Conclusions Tail-suspension can successfully simulate microgravity environment and duplicate osteoporosis model. Physiological loading can effectively prevent the emergence and development of microgravity-induced osteoporosis, while overloading can also counter microgravity-induced osteoporosis, but the results have no significant differences.