BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in infants and the elderly. Currently, there are no approved RSV-specific therapeutic small molecules available. Amentoflavone (AMF) is a well-known biflavone occurring in many plant spp with potential antiviral activities against RSV. However, the underlying mechanism remains unclear. PURPOSE: In this study, we aimed to investigate the potential of AMF in treating RSV-induced lung inflammation injury, along with the mechanism underlying its effects, focusing on the roles of gut microbiota and innate immune response. STUDY DESIGN AND METHODS: Nasal inoculation of RSV was performed on BALB/C mice to induce lung inflammation injury, followed by treatments with AMF and the positive control drug ribavirin. In addition, the mice were treated with antibiotics for 14 days to eliminate the gut microbiota and establish a model of pseudo-sterile gut, before receiving AMF drug intervention. Various techniques, including histological assays, TCID50 assays, immunofluorescence staining, immunohistochemical staining, qRT-PCR, western blotting, untargeted metabolomics, targeted metabolomics, molecular docking simulation, and molecular dynamics simulation, were utilized to determine the potential mechanisms underlying AMF's therapeutic effects in RSV-induced lung inflammation injury, as well as the role of gut microbiota in this process. RESULTS: AMF effectively inhibited RSV replication, reduced virus titer, and alleviated RSV-induced lung inflammation injury. AMF exerted antiviral effects and protected lung tissue from RSV damage by activating innate immune signaling pathways. Specifically, AMF enhanced the RIG-I-MAVS signaling pathway, leading to increased release of type I interferons (IFNs). These type I IFNs interact with the interferon receptor (IFNAR) to induce the expression of interferon-stimulated genes (ISGs), which are crucial for inhibiting RSV replication. In addition, we found that AMF was metabolized by gut microbiota to produce desaminotyrosine (DAT), a compound that interacts with IFNAR and increases the levels of ISG products. Upon the elimination of gut microbiota, DAT levels were significantly reduced, decreasing the effectiveness of AMF in the treatment of RSV-induced lung inflammation injury. This study uniquely reveals multifaceted antiviral strategy of AMF, directly engaging the RIG-I-MAVS pathway while also utilizing gut microbiota metabolism to generate the IFNAR agonist DAT, representing a novel interplay between the compound, host immunity, and the microbiome. Furthermore, our metabolomic data suggested AMF significantly modulates host metabolic pathways, potentially contributing to the enhanced antiviral state. CONCLUSION: The effects of AMF in treating RSV-induced lung inflammation injury are achieved through the activation of innate immune responses and modulation of host-microbiota metabolic interactions. Based on its novel mechanism involving concurrent direct immune activation and microbiota-dependent metabolic potentiation of IFN signaling, AMF demonstrates significant activity against RSV and can be considered a potential therapeutic agent. Furthermore, gut microbiota plays a crucial, mechanistically defined role in this antiviral process and is an essential component of the antiviral effect of AMF, highlighting the host-metabolism-immunity axis as a promising therapeutic target.