Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS). MD-2 is associated with TLR4 and imparts LPS responsiveness to it. Little is known, however, as to whether MD-2 directly regulates LPS recognition by TLR4. To address the issue, we took advantage of a species-specific pharmacology of lipid IVa, an analogue of lipid A. Lipid IVa acted agonistically on mouse (m) TLR4/MD-2 but not on human (h) TLR4/MD-2. Lipid IVa antagonized the agonistic effect of lipid A on hTLR4/MD-2. We examined the chimeric complex consisting of mTLR4 and hMD-2 to ask whether species specificity is conferred by TLR4 or MD-2. hMD-2 was clearly distinct from mMD-2 in the way of influencing LPS recognition by mTLR4. hMD-2 conferred on mTLR4 responsiveness to lipid A but not to lipid IVa. Moreover, lipid IVa acted as a lipid A antagonist on mTLR4 that is associated with hMD-2. Collectively, MD-2 directly influences the fine specificity of TLR4.