Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-beta. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-beta induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-alpha induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-beta and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKK epsilon-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-beta induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-beta. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKK epsilon-mediated induction of IRF3 and concomitant IFN-beta and RANTES production. The Journal of Immunology, 2011, 186: 2514-2522.