Pellino family proteins are E3 ubiquitin ligases that can regulate members of the IL-1 receptor-associated kinase (IRAK) family. This study shows that Pellino3 negatively regulates type I interferon (IFN) expression following Toll-like receptor 3 (TLR3) activation. The authors generated Pellino3-deficient mice and found that macrophages from these animals produced increased levels of IFN following TLR3 stimulation but responded in a similar manner to wild-type macrophages when treated with other TLR agonists. Notably, the enhanced IFN response of Pellino3-deficient mice promoted protective immunity to encephalomyocarditis virus (ECMV). Pellino3 negatively regulated TLR3 signalling by associating with and ubiquitylating TNF receptor associated factor 6 (TRAF6) — this blocked IFN induction by preventing TRAF6-mediated ubiquitylation and activation of interferon regulatory factor 7 (IRF7). Activation of TLR3 upregulated Pellino3 expression, suggesting that this is an autoregulatory pathway that controls type I IFN induction.