Protein ubiquitination is a reversible reaction, in which the ubiquitin chains are deconjugated by a family of deubiquitinases (DUBs). The presence of a large number of DUBs suggests that they likely possess certain levels of substrate selectivity and functional specificity. Indeed, recent studies show that a tumor suppressor DUB, cylindromatosis (CYLD), has a predominant role in the regulation of NF-κB, a transcription factor that promotes cell survival and oncogenesis. NF-κB activation involves attachment of K63-linked ubiquitin chains to its upstream signaling factors, which is thought to facilitate protein–protein interactions in the assembly of signaling complexes. By deconjugating these K63-linked ubiquitin chains, CYLD negatively regulates NF-κB activation, which may contribute to its tumor suppressor function. CYLD also regulates diverse physiological processes, ranging from immune response and inflammation to cell cycle progression, spermatogenesis, and osteoclastogenesis. Interestingly, CYLD itself is subject to different mechanisms of regulation.