Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.