The induction of beta-interferon (IFN-beta) is a key anti-viral response to infection by RNA viruses. Virus-induced expression of IFN-beta requires the co-operative action of the transcription factors IRF-3/7, NF-kappa B, and ATF-2/c-Jun on the IFN-beta promoter leading to the orderly recruitment of chromatin remodeling complexes. Although viruses strongly activate NF-kappa B and promote its binding to the IFN-beta promoter, recent studies have indicated that NF-kappa B is not essential for virus-induced expression of IFN-beta. Herein, we examined the role of NF-kappa B in regulating IFN-beta expression in response to the viral-sensing Toll-like receptor 3 (TLR3). Intriguingly pharmacological inhibition of the NF-kappa B pathway augments late phase expression of IFN-beta expression in response to TLR3 stimulation. We show that the negative effect of NF-kappa B on IFN-beta 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-beta promoter and inhibits the binding of IRF7 to the latter. Evidence is also presented showing that the NF-kappa B subunits c-Rel and RelB are the likely key drivers of these negative effects on IFN-beta expression. 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-beta expression.