The transcription factor NF-kappaB regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-kappaB are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-kappaB signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-kappaB essential modulator, also known as IkappaB kinase gamma). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-kappaB by TNF-alpha and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-kappaB signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-kappaB-dependent induction of CYLD by TNF-alpha and NTHi. These findings provide novel insights into the autoregulation of NF-kappaB activation.