Interleukin-1 receptor associated kinase (IRAK)-1 is a serine/threonine kinase which has previously been proposed to act as a negative regulator of type I Interferon (IFN) production downstream of TLR3 (1). This study has been able not only to confirm this finding, but also uncovered multiple mechanisms by which IRAK1 regulates the TRIF-dependent pathway. This work has also revealed novel roles for the non-canonical kinases IKKε and TBK1. Kinase-active IRAK1 has been shown to limit the activation of TBK1, JNK and p38 MAPK in an IKKε-TAK1-dependent manner. In particular, IRAK1 was found to be required for the activation of IKKε, and the mutual phosphorylation of the two kinases has been demonstrated in vitro. Whilst IKKε limits the pathway to NF-κB, TBK1 has been shown to be involved in the activation of the canonical IKKs. This work also revealed that the kinase activities of IRAK1 and IKKε are required for the activation of Erk1/2 downstream of TLR3. Furthermore, IRAK1 has also been found to phosphorylate IRF3 in vitro, and limit the activation of the TBK1-IRF3 signalling to type I IFN also in an IKKε-independent manner, indicating the existence of at least a dual mechanism of inhibition of the TLR3 signalling cascade. IL-27 is a cytokine found to be a key regulator of inflammatory responses (2–4). This study has shown that IRAK1 controls IL-27 production downstream of TLR3 and TLR4 in a TBK1-dependent manner. Increased IRF1 and STAT1 levels were found in the IRAK1-deficient compared to the wild-type BMDM. Overall, this work provides novel insight into the numerous functions of IRAK1 in modulating the TLR3 pathway. Moreover, this study identifies for the first time a TLR signalling molecule, namely IRAK1, as a negative regulator of IL-27 production downstream of TLR3 and TLR4.