Abstract: Objective: To evaluate improvement of sleep function and mechanism of Yanwo Ejiao compound in mice. Methods: Balb-c mice were randomly assigned to five groups: A blank control group, a positive drug group, and three Yanwo Ejiao compound groups receiving low (5 g/kg), medium (10 g/kg), and high (20 g/kg) doses. The compound was administered once daily for 30 consecutive days. Sleep improvement was assessed by direct sleep test, subthreshold dose hypnosis test with pentobarbital sodium, pentobarbital sodium-induced sleep time extension test, and barbital sodium sleep latency test. Additionally, network pharmacology methods were employed to preliminarily investigate the compound's potential mechanism. To validate the findings, enzyme-linked immunosorbent assay kits were used to measure brain levels of 5-hydroxytryptamine (5-HT), dopamine (DA), tryptophan hydroxylase (TPH), and γ-aminobutyric acid (GABA). Results: Yanwo Ejiao compound did not induce mice directly into a hypnotic state, but significantly improved sleep quality in mice. Compared to the blank control group, the low, medium, and high-dose groups increased the sleep rate by 50%, 70%, and 80%, respectively. Three Yanwo Ejiao compound groups significantly prolonged pentobarbital sodium-induced sleep time (P<0.01). The medium and high-dose groups also significantly reduced sleep latency (P<0.05 or P<0.01). Network pharmacology analysis suggested that the compound improved sleep by modulating neurotransmitter synaptic pathways, including those of 5-HT, GABA, and DA. Experimental data confirmed that, compared to the control group, the medium and high-dose group significantly increased TPH levels (P<0.01 or P<0.05) and three compound groups elevated 5-HT and GABA levels in brain tissue (P<0.01 or P<0.05). Besides, three Yanwo Ejiao compound groups significantly decreased DA content (P<0.01). Conclusion: Yanwo Ejiao compound exerts a notable sleep-improvement effect in mice, primarily through the regulation of synaptic pathways involving neurotransmitters such as 5-HT, GABA, and DA.