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.