IκBζ (inhibitor of NF-κB (nuclear factor κB) ζ) is a nuclear protein induced upon stimulation of toll-like receptors (TLRs) and interleukin-1 receptor. Induced IκBζ, especially its C-terminal ankyrin repeat domain (ARD), interacts with NF-κB in the nucleus, where it regulates the transcriptional activity of target genes. Recent studies have shown that human ARD of IκBζ binds with p50/p65 heterodimer and inhibits the transcription of NF-κB regulated genes, whereas mouse ARD of IκBζ binds with p50/p50 homodimer and exhibits transcriptional activation activity. Since human and mouse IκBζ ARD are identical, it is unclear how IκBζ can be a positive and negative regulator of NF-κB-mediated transcription. Therefore, we generated a structural model of IκBζ ARD and constructed a detailed molecular dynamics (MD) simulation of IκBζ in explicit solvent to investigate ARD flexibility. In addition, we used molecular docking to screen for potential sites of interaction between IκBζ and the p50/p65 heterodimer and IκBζ and the p50/p50 homodimer. The docking experiments revealed that the binding of IκBζ ankyrin repeats with the p50/p65 N-terminal DNA binding domain prevents NF-κB-mediated transcriptional activation. Furthermore, the IκBζ–p50 homodimer complex, which lacks Pro, Glu (and Asp), Ser and Thr (PEST motif), facilitated gene expression. These two different binding schemes of IκBζ may be responsible for its opposite function, which is consistent with the currently available biochemical data. Moreover, our data implicate structurally highly flexible ARD residues as the prime contributors to this dual function. Copyright © 2010 John Wiley & Sons, Ltd.