IκB-ζ, an essential inflammatory regulator, is specifically induced by Toll-like receptor ligands or interleukin (IL)-1β by post-transcriptional activation mediated via a 165-nucleotide element in IκB-ζ mRNA. Here, we analyzed the Toll-like receptor–IL-1 receptor signaling components involved in the post-transcriptional regulation of IκB-ζ with mutated estrogen receptor [ER(T2)] fusion proteins. Upon 4-hydroxytamoxifen treatment, the ER(T2) fusion proteins with IL-1 receptor-associated kinase (IRAK)1 and IRAK4 elicited specific activation of a reporter gene for the post-transcriptional regulation of IκB-ζ. The tumor necrosis factor receptor-associated factor (TRAF)6–ER(T2) protein activated nuclear factor-κB, but not post-transcriptional regulation, indicating that activation of IRAK1/4, but not of TRAF6, is sufficient to activate the 165-nucleotide element-mediated post-transcriptional mechanism. Interestingly, the post-transcriptional mechanism was not activated in TRAF6-deficient cells, indicating an essential role for TRAF6. Thus, the signaling pathway leading to nuclear factor-κB activation and the post-transcriptional activation bifurcates at IRAK1, suggesting a new pathway activated by IRAK1.