Mammalian Pellino isoforms are phosphorylated by IRAK1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. Here we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDM are stimulated with LPS or poly(I:C). This induction occurs via a TRIF-dependent, IRAK-independent pathway and is prevented by inhibition of the IKK-related protein kinases, TBK1 and IKKepsilon. Pellino 1 is not induced in IRF3-/- BMDM, and is only reduced slightly in type 1 interferon receptor-/- BMDM, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKepsilon as bait, and show that IKKepsilon/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by inhibition of IKKepsilon/TBK1 and is reversed by phosphatase treatment. Thus IKKepsilon/TBK1 mediate the activation of Pellino 1's E3 ligase activity as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.