The degradation profile of mulberrin (Mul) in the oral-gastrointestinal tract and its inhibitory effects on Lachnospira bacteria, along with associated metabolic regulatory mechanisms, were herein elucidated. In vitro simulated digestion models coupled with high-performance liquid chromatography were employed to quantitatively analyze the dynamic changes and digestive rates of Mul in gastrointestinal environments. Five mass concentration gradients of Mul were administered to Lachnospira to assess dose-dependent effects. Metabolites derived from Lachnospira biotransformation of Mul were collected at three time points and systematically analyzed using untargeted metabolomics. After 2 h in simulated gastric conditions, 71.50% of Mul was digested, while complete degradation (100%) was observed after 2 h in simulated intestinal environments. A significant dose-dependent inhibitory effect on Lachnospira was demonstrated, with an inhibition rate of 89.35% at the highest concentration (1 000 μg•mL−1). Distinct metabolic profiles were observed between Mul-treated and blank control groups, wherein Lachnospira dynamically converted Mul into characteristic metabolites, including parvisoflavone B, kuwanon K, and morusinol. Mul exhibited gradient digestion in the oralgastrointestinal system with its inhibitory efficacy being concentration dependent. Additionally, Mul modulated lipid metabolic homeostasis by regulating Lachnospira abundance, inducing characteristic metabolite production, and significantly altering linoleic acid (P<0.000 1), arachidonic acid (P<0.05), and caffeine (P<0.05) metabolic pathways. This study provides key mechanistic insights for developing flavonoid-based microbiota-targeted anti-obesity strategies.