Polygoni Multiflori Caulis (PMC) has been traditionally served as a functional medicine due to its various biological activities, but a lack of researches focused on the sleep-enhancing effects of flavonoids from PMC. In this study, the total flavonoids (PMCF) were isolated from PMC. In vitro antioxidant experiments suggested that PMCF exhibited remarkable antioxidant capacity, as evidenced by its scavenging activity on several radicals and reducing power. Subsequently, PMCF was given to insomnia mice via intragastric administration. In behavior test, PMCF exhibited sleep-enhancing effect through suppressing depression, shortening the sleep latency time while prolonging the duration sleep time in insomnia mice. To clarified the mechanism, the neurotransmitter receptors, cytokines and hormone in brain were investigated. PMCF exhibited significant sleep promoting effect by up-regulating the expression levels of hormone-related genes, and producing stimulation on the immune system by increasing interleukin-1β mRNA expression. In addition, PMCF-mediated sleep promotion accompanied the recovery of 5-hydroxytryptamine 1 A (5-HT1A), γ-aminobutyric acid A receptor α2 (GABAARα2) and GABAA receptor α3 (GABAARα3) mRNA expression, which was more effective in the hypothalamus. Collectively, all these data implied that PMCF might produce beneficial activities in relieving sleep disturbances via the antioxidant pathway, as well as the immune, serotonergic and GABAergic systems.