Engagement of the interleukin-1 (IL-1) and Toll-like receptors triggers mitogen-activated protein kinase (MAPK) pathways and activation of transcription factors such as NFkappaB and AP-1. Recent studies have identified members of the Pellino protein family as novel mediators in mediating activation of these pathways. However, no evidence has been presented to date to suggest a role for the Pellino proteins in activation of the p38 MAPK pathway. We demonstrate herein that Pellino3 is a strong activator of p38 MAPK. RNA interference was used to reveal a physiological role for Pellino3 in the IL-1 pathway leading to activation of p38 MAPK. A series of N-terminal truncation and point mutants of Pellino3 were generated and tested for their ability to activate p38 MAPK in an effort to map sites of protein interaction important for p38 MAPK activation. In this way we show that the binding of Pellino3 to IL-1 receptor-associated kinase 1 coincides with its ability to promote p38 MAPK activation. TRAF-6 and transforming growth factor-beta-activating kinase 1 are shown to act as downstream mediators of the activation of p38 MAPK by Pellino3. Finally we confirm the functional consequences of the activation of p38 MAPK by Pellino3 by demonstrating that Pellino3 promotes translocation of the p38 substrate, MAPK-activated protein kinase2, from the nucleus to the cytoplasm and activates the transcription factor CREB in a p38 MAPK-dependent manner. Our study not only identifies Pellino3 as a novel upstream regulator of the p38 MAPK pathway but also probes the mechanistic basis underlying the ability of Pellino3 to promote activation of this pathway.