The induction of Î²-interferon (IFN-Î²) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-Î² requires the co-operative action of the transcription factors IRF-3/7, NF-Î²B, and ATF-2/c-Jun on the IFN-Î² promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-Î²B and promote its binding to the IFN-Î² promoter, recent studies have indicated that NF-ÎºB is not essential for virus-induced expression of IFN-Î². Herein, we examined the role of NF-ÎºB in regulating IFN-Î² expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-ÎºB pathway augments late phase expression of IFN-Î²expression in response to TLR3 stimulation. We show that the negative effect of NF-ÎºB on IFN-Î² expression is dependent on the induction of the transcriptional repressor protein YinYang1. We demonstrate that the TLR3 ligand polyriboinosinic: polyribocytidylic acid (poly(I:C)) induces expression and nuclear translocation of YinYang1 where it interacts with the IFN-Î²promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-ÎºB subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-Î²expression. These findings thus highlight for the first time a novel self-regulatory mechanism that is employed by TLR3 to limit the level and duration of IFN-Î² expression. Â© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.