Studies suggest that mangiferin (MAF) has good therapeutic effects on chronic bronchitis and hepatitis. Also, it is one of the antiviral ingredients in Anemarrhena asphodeloides Bunge. However, its effect on the LPS-induced inflammation and intestinal flora during sepsis remains unclear yet. In the present study, LPS-stimulated inflammation RAW264.7 cells and LPS-induced sepsis mice were used to evaluate the efficacy of MAF in vitro and in vivo. 16S rDNA sequencing was performed to analyze the characteristics of intestinal flora of the sepsis mice. It has been demonstrated that MAF (12.5 and 25 mu g/mL) significantly inhibited protein expressions of TLR4, MyD88, NF-kappa B, and TNF-alpha in the LPS-treated cells and reduced the supernatant TNF-alpha and IL-6 levels. In vivo, MAF (20 mg/kg) markedly protected the sepsis mice and reduced the serum TNF-alpha and IL-6 levels. Also, MAF significantly downregulated the protein expressions of TLR4, NF-kappa B, and MyD88 in the livers. Importantly, MAF significantly attenuated the pathological injuries of the livers and small intestines. Further, MAF significantly increased proportion of Bacteroidota and decreased the proportions of Firmicutes, Desulfobacterota, Actinobacteriota, and Proteobacteria at phylum level, and it markedly reduced the proportions of Escherichia-Shigella, Pseudoalteromonas, Staphylococcus at genus level. Moreover, MAF affects some metabolism-related pathways such as citrate cycle (TCA cycle), lipoic acid metabolism, oxidative phosphorylation, bacterial chemotaxis, fatty acid biosynthesis, and peptidoglycan biosynthesis of the intestinal flora. Thus, it can be concluded that MAF as a treatment reduces the inflammatory responses in vitro and in vivo by inhibiting the TLR4/ MyD88/NF-kappa B pathway, and corrects intestinal flora imbalance during sepsis to some degree.