The MD-2/TLR4 complex provides a highly robust mechanism for recognition and response of mammalian innate immunity to Gram-negative bacterial endotoxins. Despite overall close structural and functional similarity, human (h) and murine (m) MD-2 show several species-related differences, including the ability of hMD-2, but not mMD-2, to bind endotoxin (E) in the absence of TLR4. Wild-type mMD-2 can support TLR4-dependent cell activation by E only when mMD-2 and mTLR4 are coexpressed in the same cell. However, replacement of Glu122, Leu125, and/or Asn58 of mMD-2 with the corresponding residues (lysines) of hMD-2 was sufficient to yield soluble extracellular MD-2 that reacted with monomeric E . sCD14 complex to form extracellular monomeric E . MD-2 that activated cells expressing TLR4 without MD-2. Moreover, in contrast to wild-type mMD-2, double and triple mMD-2 mutants also supported E-triggered signaling in combination with human TLR4. Conversely, a K125L mutant of hMD-2 reacted with E . CD14 and activated TLR4 only when coexpressed with TLR4, and not when secreted without TLR4. These findings reveal novel roles of lysines 122, 125, and 58 in human MD-2 that contribute to the functional differences between human and murine MD-2 and, potentially, to differences in the sensitivity of humans and mice to endotoxin.