Objective To observe the different characteristics of spontaneous activitiy after simulated weightlessness 21 days in rats, aimed to provide a evaluation method for space weightlessness induced function change in human beings and to provide a reference for researches on the astronauts protective measures. Methods 30 Wistar male rats were randomly divided into three groups, the control group, the sham tail-suspended hindlimb unloading group (the sham group), the tail-suspended hindlimb unloading group (the suspending group), ten animals in each group. All animals were placed in the simulated space flight environmental equipment which has a real-time monitor system for 21 days. During the 21 days, the intake of water, food and the body weight were measured every week. Meanwhile, five independent activity data were collect every day, for example, morning(8:00am～ 12:00am), afternoon(2:00pm～6:00pm), daytime(8:00am～8:00pm),night(8:00pm～8:00am),and whole day (8:00am～8:00am). Results The spontaneous activity of normal rats in the control group between morning and afternoon had no significant difference, but it is significantly between night and daytime. The movement time and distance in night are 2-3 times than that of the daytime. After 10 days of tail suspending, the circadian rhythm was disordered, and the spontaneous activity in day and night become more similar in rats of the suspending group. Because of the individual difference, the spontaneous activity is not stable at the first 10 days in rats of the sham group, but after 10 days, it become close to the control group. Conclusion Rat is nocturnal animal and sleeps in the daytime, the spontaneous activity in night is 2-3 times as compared with the daytime. The sham tail-suspended hindlimb unloading 21 days can not influence the circadian rhythm in rats. Tail suspending 21 days will caused to the disappearance in the circadian rhythm in rats.