The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-κB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCζ. Ajuba recruits TRAF6 to p62 and in vitro activates PKCζ activity and is a substrate of PKCζ. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-κB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-κB activity following IL-1 stimulation. We propose that Ajuba is a new cytosolic component of the IL-1 signaling pathway modulating IL-1-induced NF-κB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex.