The NF-κB family of transcription factors is a central regulator of chronic inflammation. The phosphorylation of IκB proteins by the IκB kinase (IKK) complex (IKKα, IKKβ, and NF-κB essential modulator or NEMO) is a key step in NF-κB activation. Peptides corresponding to the NEMO binding domain (NBD) of IKK blocks NF-κB activation without inhibiting basal NF-κB 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-κB reporter system, 8K-NBD dose-dependently inhibits TNF-induced NF-κB activation. Furthermore, 8K-NBD inhibited nuclear translocation of NF-κB family members. In NF-κB EGFP knock-in mice, 8K-NBD inhibited LPS-activated NF-κB (EGFP activity) in the ileum but did not inhibit basal NF-κB in Peyer’s patches. IL-10−/− mice treated systemically with 8K-NBD demonstrate amelioration of established colitis, decreased NF-κB activation in the lamina propria, and a reduction in spontaneous intestinal IL-12 p40, TNF, IFN-γ, 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.