The interactions of Toll-like receptor 4 (TLR4) with competitive inhibitors were investigated by a combined ligand-based and target-based approach. Firstly, the ligand-based pharmacophore model of the reported TLR4 inhibitors was constructed by utilizing the common feature method, which included three hydrophobic groups and a hydrogen bond receptor. The Schrödinger software suite glide module was used to dock inhibitors with proteins and verify the importance of these four interaction points from the target level. Then, molecular dynamics, alanine scanning mutagenesis, and binding free energy calculation were used to identify the key amino acids in the binding mode. In addition, blind docking proved that the TLR4 inhibitor does not bind to TLR4 itself like other TLR family proteins. Based on this, we also screened a class of sesquiterpene coumarins which possibly have TLR4 inhibitory activity and will conduct a detailed study later. Together, this study revealed the interactions between TLR4 protein and its competitive inhibitors, which shed light on better access for developing its novel inhibitors.