Objective To investigate the influence of hyper-gravity environment on biomechanical properties and morphological structure of bone tissues, so as to provide theoretical support for safe flight training of astronauts and pilots and determination of the maximum tolerance value of hyper-gravity. Methods The hyper-gravity animal model was established by using the self-designed hyper-gravity loading platform. The loading mode was the ventral-dorsal direction which was subjected to centripetal acceleration of 5 g, 10 g, and 20 g, respectively (45 s, once a day, 5 d/ week, for 4 weeks). Instron 5865 material testing machine was used to perform three- point bending test on in vitro tibia, and the influence of hyper-gravity environment on mechanical properties of bone tissues was analyzed by measuring changes in mechanical parameters. Dual-energy X-ray absorptiometry, micro-CT scanning and bone tissue staining were utilized to test changes in bone mineral contents, bone mineral density, and to observe the influence of hyper-gravity environment on bone morphology. Results Hyper-gravity loading could reduce fracture stress and elastic modulus of the tibia in mice. The bone volume/ total volume (BV/ TV), trabecular thickness (Tb. Th) and trabecular number (Tb. N) were significantly decreased, and the trabecular arrangement was disordered under hyper-gravity environment. Conclusions The effect of hyper-gravity on biomechanical properties and morphological structure of bone tissues is related to the intensity of hyper- gravity. Low-strength ( 5 g) overload can reduce Tb. Th, but has little effect on its mechanical properties. Moderate ( 10 g) and high-strength ( 20 g) overload can significantly trigger bone loss and decrease bone mechanical properties. With the increase of hyper-gravity load, physiological regulation of the body can not prevent the decline of bone mass and mechanical properties.