Objective To study the pharmacokinetics of melatonin in rat plasma under conditions of normal gravity and simulated microgravity. Methods First, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) method was established to determine the concentration of melatonin in rat plasma. The simulated microgravity model of SD rats was established by 21 days of tail suspension. The rats in the normal gravity group were used as the control. After 21 days, the rats in both groups were administered melatonin (0. 27 mg / kg) by single gavage. Blood was taken from the jugular vein at 5, 10, 15, 20, 30, 40 min and 1, 1. 5, 2, 4 and 6 hours after administration, and the plasma was separated. The concentration of melatonin in the rat plasma was analyzed by the above-established UPLC-MS /MS method and the pharmacokinetic parameters were calculated. Results Within the concentration range of 0. 1 ~ 50 ng /mL, the linear relationship of melatonin in plasma was good. The relative standard deviation (RSD%) for both intra-day and inter-day precision was < 10. 99%. The extraction recoveries of low, medium, and high concentrations (0. 1, 25, and 50 ng / mL) were between 99. 3% and 115. 3%, and there was no obvious matrix effect. Compared with the normal gravity group, the pharmacokinetic parameters of melatonin in the plasma of simulated microgravity rats changed significantly. There were significant differences in time to reach maximum concentration ( T_max ), area under the plasma concentration-time curve (AUC_{0→6 h} ) , plasma clearance (CL), and mean residence time ( MRT_{0→6 h} )(P< 0. 05). The T_max, AUC_{0→6 h} , CL, and MRT_{0→6 h} values in the plasma of simulated microgravity rats were 1. 6, 3. 1, 0. 34, and 1. 56 times those of the normal gravity control rats, respectively. The bioavailability of melatonin in the microgravity rats was 312. 5% that of the normal gravity rats. Conclusions Compared with normal gravity, the pharmacokinetic characteristics of melatonin changed significantly after 21 days of simulated microgravity.