Abstract: Objective: To explore the effect of water extract of Ziziphi Spinosae Semen, Poria Cocos and Codonopsis Radixon (1:1:1) on sleep improvement in mice and its potential mechanism. Methods: 60 male KM mice were randomly divided into 6 groups: normal group, solvent control group, Ziziphi Spinosae Semen-Poria cocos-Codonopsis Radixon low, medium, high dose group (14, 28, 56 g/kg), positive drug group (diazepam, 2.5 mg/kg), autonomous activity experiments and pentobarbital induced sleep experiments were performed; another 60 KM mice were tested for insomnia induced by p-chlorphenylalanine, It was divided into normal group, model group, Ziziphi Spinosae Semen-Poria cocos-Codonopsis Radixon low, medium, high dose group, positive drug group, 10 in each group, gavage dose of 10 mL/(kg·d), continuous administration for 30 d. The sleep latency and time were recorded by sodium pentobarbital, and the effect of water extract on promoting sleep and prolonging sleep time was evaluated. By recording the autonomous activity of mice, analyzing the score of level and the distance of activity, we evaluated the effect of water extract on the improvement of PCPA induced insomnia mice and the effect on normal mice. Contents of 5-HT and TPH in hypothalamus and cortex, Glu and GABA in hippocampus and cortex of mice were detected. Results: The extract could significantly reduce the sleep latency, prolong the sleep time, reduce the autonomous activity ability of insomnia mice, and reduce the level score and activity distance of normal mice (P < 0.05, P< 0.01); enhance the activity of 5-HT synthesis key enzyme TPH, increase the content of 5-HT in hypothalamus (P< 0.05); regulate the content of hippocampal Glu and GABA and maintain the Glu/GABA balance (P< 0.05). Conclusion: The water extract of Ziziphi Spinosae Semen, Poria Cocos and Codonopsis Radixon can improve sleep, and the mechanism is related to regulating the serotonin system and Glu/GABA homeostasis in the mouse brain.