The NF-kappaB family of transcription factors is a central regulator of chronic inflammation. The phosphorylation of IkappaB proteins by the IkappaB kinase (IKK) complex (IKKalpha, IKKbeta, and NF-kappaB essential modulator or NEMO) is a key step in NF-kappaB activation. Peptides corresponding to the NEMO binding domain (NBD) of IKK blocks NF-kappaB activation without inhibiting basal NF-kappaB activity. In this report, we determined the effects of the IKK inhibitor peptide (NBD) in a model of spontaneously occurring chronic murine colitis, the IL-10-deficient (IL-10(-/-)) mouse. Using a novel cationic peptide transduction domain (PTD) consisting of eight lysine residues (8K), we were able to transduce the NBD peptide into cells and tissues. In a NF-kappaB reporter system, 8K-NBD dose-dependently inhibits TNF-induced NF-kappaB activation. Furthermore, 8K-NBD inhibited nuclear translocation of NF-kappaB family members. In NF-kappaB(EGFP) knock-in mice, 8K-NBD inhibited LPS-activated NF-kappaB (EGFP activity) in the ileum but did not inhibit basal NF-kappaB in Peyer's patches. IL-10(-/-) mice treated systemically with 8K-NBD demonstrate amelioration of established colitis, decreased NF-kappaB activation in the lamina propria, and a reduction in spontaneous intestinal IL-12 p40, TNF, IFN-gamma, and IL-17 production. These results demonstrate that inhibitors of IKK, in particular a PTD-NBD peptide, may be therapeutic in the treatment of inflammatory bowel disease.