Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-κB (NF-κB) activation. Endotoxin-depleted hLF induces NF-κB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-κB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-κB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF-κB activation in myeloid differentiating factor 88 (MyD88)-dependent signaling pathways, while the role of TRAF6 in the MyD88-independent signaling pathway has not been clarified extensively. When we examined the hLF-dependent NF-κB activation in MyD88-deficient MEFs, delayed, but remarkable, NF-κB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88-dependent and MyD88-independent pathways are involved. Indeed, hLF fails to activate NF-κB in MEFs lacking Toll-like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide-induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4-mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide-dependent TLR4 activation, probably through its polypeptide moiety.