Abstract Prior exposure to LPS induces endotoxin tolerance that impairs TLR4-elicited pro-inflammatory cytokines without inhibiting anti-inflammatory or anti-microbial mediators, and is important for pathogenesis of the anti-inflammatory response syndrome and secondary infections in septic patients. The impact of endotoxin tolerance on the earliest TLR4 signaling events is largely unknown. We report that endotoxin tolerization impairs LPS-mediated activation of IRAK4 and TAK1 in human monocytes and THP1 cells. Overexpression of wild-type and mutant ubiquitin variants and the use of antibodies discriminating K48- vs. K63-linked polyubiquitin chains revealed that endotoxin tolerization compromises LPS-induced K63-linked, but not K48-linked polyubiquitination of IRAK1 and TRAF6. Diminished K63-linked polyubiquitination of IRAK1 and TRAF6 correlated with compromised LPS-inducible IRAK1-TRAF6 and IRAK1-IKKγ complex assemblies observed in endotoxin-tolerant cells. Alterations in these proximal signaling events manifested by LPS-tolerant cells correlated with increased expression of A20, an essential de-ubiquitination enzyme that targets TRAF6 to remove K63-linked polyubiquitin chains. Thus, deficiencies in LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6 and IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased expression of A20 represent new critical molecular determinants of endotoxin tolerance.