Targeting Toll‐like receptor 4/myeloid differentiation factor 2 (TLR4/MD2) signaling is regarded as a potential strategy for treating inflammatory diseases. Saponaria officinalis L. is rich in saponin, which include quillaic acid, gypsogenin, saponarin, and hederagenin. We evaluated the pharmacological activity of a Saponaria officinalis extract in THP‐1 derived macrophages and RAW264.7 macrophages. TLR4/MyD88 complex formation and downstream signals were investigated by co‐immunoprecipitation (Co‐IP). In silico docking simulation was conducted to predict binding scores and perform 3D modeling of saponarin‐TLR4/MD2 complex. A hexane fraction of Saponaria officinalis (SH) and fr.1 (a sub‐fraction 1 of SH) inhibited mitogen‐activated protein kinase (MAPK) signaling, nuclear factor kappa b (NF‐κB) activity, cytokine production, and the expressions of marker genes specific for M1 polarization. The inhibitory effects of fr.1 and saponarin on TLR4/MyD88 complex formation were observed by western blotting TLR4 co‐immunoprecipitated proteins. Saponarin and fr.1 markedly attenuated LPS‐induced inflammatory cytokines, thus reducing mortality and morphological abnormality in zebrafish larvae. Finally, docking simulation revealed that saponarin can directly interact with TLR4/MD2 complex to inhibit downstream signalings. Our findings suggest that saponarin reduces downstream inflammatory response by disrupting TLR4/MD2 complex and blocking MyD88‐dependent inflammatory signaling.