This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on I kappa B kinase (IKK) activation, where divergent NF-kappa B-activating stimuli converge. FAF1 interacts with IKK beta in response to proinflammatory stimuli (such as tumor necrosis factor-a, interleukin-1 beta, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKK beta with FAF1 affected the IKK heterocomplex (IKK alpha/beta) and homocomplex (IKK alpha/alpha, IKK beta/beta) formations and attenuated IKK gamma recruitment to IKK beta. Overexpression of FAF1 reduced the level of IKK,13 activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKK beta. Taken together, FAF1 robustly suppresses NF-kappa B activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-kappa B p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544-2549). Such redundant suppression would prevent inadvertent activation of the NF-kappa B pathway.