Macrocyclic peptides are capable of binding to flat protein surfaces such as the interfaces of protein-protein interactions with antibody-like affinity and specificity, but generally lack cell permeability in order to access intracellular targets. In this work, we designed and synthesized a large combinatorial library of cell-permeable bicyclic peptides, in which the first ring consisted of randomized peptide sequences for potential binding to a target of interest, while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the I kappa B kinase alpha/beta (IKK alpha/beta)-binding domain of NF-kappa B essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKK beta interaction with low mu M IC50 values. Further optimization of one of the hits led to a relatively potent and cell permeable NEMO inhibitor (IC50 = 1.0 mu M), which selectively inhibited canonical NF-kappa B signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells. The inhibitor provides a useful tool for investigating the biological functions of NEMO/NF-kappa B and a potential lead for further development of a novel class of anti-inflammatory and anticancer drugs.