Sepsis, a life-threatening condition driven by dysregulated inflammation, remains a major clinical challenge due to high mortality rates and limited therapeutic options. This study investigates the anti-inflammatory properties of Leucoside, a natural flavonoid isolated from tea seed extract, and its potential as a therapeutic agent for sepsis. Using a bacterial infection-induced septic mouse model and lipopolysaccharide (LPS)-activated macrophages, we demonstrated that Leucoside significantly improves survival rates, reduces hypothermia, and attenuates organ damage by suppressing systemic inflammation. Mechanistically, network pharmacology and molecular docking identified Toll-like receptor 4 (TLR4) as a primary target of Leucoside. Biochemical and structural analyses revealed that Leucoside competitively binds to conserved positively charged residues in the B patch of TLR4, specifically Lys263 and Arg337, forming a spatial barrier that inhibits the formation of the myeloid differentiation protein 2 (MD2)-TLR4 complex and subsequent nuclear factor kappa-B (NF-κB) signaling. This inhibition was further validated through co-immunoprecipitation assays, which showed a reduced effect on the TLR4-MD2 complex dissociation when Lys263 and Arg337 were mutated. These findings highlight Leucoside as a novel TLR4 inhibitor with significant potential for treating sepsis and other TLR4-mediated inflammatory diseases. By elucidating its mechanism of action, this study provides a foundation for developing targeted therapies to address the unmet clinical needs in sepsis management.