Metabolic-associated fatty liver disease (MAFLD) is the most prevalent chronic liver disease globally, with no effective pharmacological treatments available for early-stage cases. Rutin, a bioactive flavonoid from Sophora japonica L., exhibits diverse pharmacological effects, but its multi-pathway mechanisms in improving MAFLD remain unclear. In this study, we employed a high-fat diet (HFD)-induced MAFLD mouse model to investigate the therapeutic effects of rutin supplementation. Rutin supplementation significantly reduced blood lipid and liver lipid levels and alleviated liver injury in MAFLD model mice. Fecal microbiota transplantation experiments revealed that rutin alleviated MAFLD by modulating the gut microbiota composition. Through 16S rRNA sequencing analysis and non-targeted metabolomics analysis of the normal control (NC), HFD and rutin groups, rutin was found to alter key species (Ruminococcus torques) and associated metabolites (e.g., 7-dehydrocholesterol, short-chain fatty acids), suggesting a mechanism involving the gut microbiota. Antibiotic treatment experiments revealed that rutin alleviates MAFLD via the blood entry pathway. Network pharmacology analysis showed that rutin can directly act on targets closely related to MAFLD development, including tumor protein p53, epidermal growth factor receptor, and prostaglandin-endoperoxide synthase 2, as well as key signaling pathways such as PI3K/AKT and MAPK. Transcriptomics analysis of the NC, HFD and rutin groups revealed that rutin may ameliorate MAFLD through PI3K/AKT and MAPK signaling pathways, which might be enhanced by the gut microbiota and blood entry pathways. In conclusion, rutin can treat MAFLD through both the gut microbiota and blood entry pathways, resulting in a synergistic effect. Our study provides a novel strategy for evaluating functional food components and offers a scientific basis for dietary flavonoid-based interventions against MAFLD.