Toll-like receptor 4 (TLR4) is crucial in the innate immune response with species-specific recognition. As a novel small-molecule agonist for mouse TLR4/MD2, Neoseptin 3 fails to activate human TLR4/MD2, while the underlying mechanism is unclear. Herein, molecular dynamics simulations were performed to investigate the species-specific molecular recognition of Neoseptin 3. Lipid A, a classic TLR4 agonist showing no apparent species-specific sensing by TLR4/MD2, was also investigated for comparison. Neoseptin 3 and lipid A showed similar binding patterns with mouse TLR4/MD2. Although the binding free energies of Neoseptin 3 interacting with TLR4/MD2 from mouse and human species were similar, protein-ligand interactions and the details of the dimerization interface were substantially different between Neoseptin 3-bound mouse and human heterotetramers at the atomic level. Neoseptin 3 binding made human (TLR4/MD2)2 more flexible than human (TLR4/MD2/Lipid A)2, especially at the TLR4 C-terminus and MD2, which drives human (TLR4/MD2)2 fluctuating away from the active conformation. In contrast to mouse (TLR4/MD2/2*Neoseptin 3)2 and mouse/human (TLR4/MD2/Lipid A)2 systems, Neoseptin 3 binding to human TLR4/MD2 led to the separating trend of the C-terminus of TLR4. Furthermore, the protein-protein interactions at the dimerization interface between TLR4 and the neighboring MD2 in the human (TLR4/MD2/2*Neoseptin 3)2 system were much weaker than those of the lipid A-bound human TLR4/MD2 heterotetramer. These results explained the inability of Neoseptin 3 to activate human TLR4 signaling and accounted for the species-specific activation of TLR4/MD2, which provides insight for transforming Neoseptin 3 as a human TLR4 agonist.