The transcriptional activation of NF-kappaB, a critical player in both physiological and pathological cellular responses to diverse cytokines, is dependent on IKK activation. Although molecular mechanisms underlying IKK activation have been well elucidated, the processes that negatively regulate IKK activity are still largely unknown. Using yeast two-hybrid screening, we have identified GbetaL as an interacting partner of IKKbeta. In this study, we demonstrate that GbetaL interacts with IKKalpha and IKKbeta in vitro and in vivo. The C-terminal WD domains of GbetaL are required for the interaction with both the kinase domain and leucine zipper domain of IKKbeta. Overexpression of GbetaL inhibits TNFalpha-induced activation of NF-kappaB signaling, while down-regulation of GbetaL expression by small interfering RNA enhances NF-kappaB activity. GbetaL constitutively interacts with IKKbeta, and this interaction is enhanced by TNFalpha treatment. GbetaL also inhibits TNFalpha-induced phosphorylation of IKKs. Taken together, these data suggest that GbetaL is involved in the negative regulation of TNFalpha-stimulated NF-kappaB signaling through a direct interaction with IKK.