Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-κB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H2O2) have antiinflammatory effects in neutrophils, including inhibition of the degradation of IκBα after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H 2O2 resulted in diminished ubiquitination of IκBα and decreased SCFβ-TrCP ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H2O2 was associated with reduced binding between phosphorylated IκBα and SCFβ-TrCP but no change in the composition of the SCF β-TrCP complex. Lipopolysaccharide-induced SCF β-TrCP ubiquitin ligase activity as well as binding of β-TrCP to phosphorylated IκBα was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H2O2 are increased. Exposure to H2O2 resulted in oxidative modification of cysteine residues in β-TrCP. Cysteine 308 in Blade 1 of the β-TrCP β-propeller region was found to be required for maximal binding between β-TrCP and phosphorylated IκBα. These findings suggest that the anti-inflammatory effects of H2O2 may result from its ability to decrease ubiquitination as well as subsequent degradation of IκBα through inhibiting the association between IκBα and SCFβ-TrCP. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.