Epidermal and mucosal epithelial cells are integral to host defense. They not only act as a physical barrier, but also utilize pattern recognition receptors, such as the Toll-like receptors (TLRs), to detect and respond to pathogens. Members of the Interferon Regulatory Factor (IRF) family of transcription factors are key components of TLR signaling as they impart specificity to downstream responses. Although IRF6 is a critical regulator of epithelial cell proliferation and differentiation, its role in TLR signaling has not previously been addressed. We show here that IRF6 is activated by IRAK1, as well as by MyD88, but not by TRIF or TBK1. Co-immunoprecipitation experiments further demonstrated that IRF6 can interact with IRAK1. Gene silencing in epithelial cells, along with gene promoter reporter assays, showed that IRAK1 mediates TLR2-inducible CCL5 gene expression, at least in part, by promoting IRF6 activation. Conversely, IRAK1 regulated CXCL8 gene expression independently of IRF6, thus identifying a molecular mechanism by which TLR2 signaling differentially regulates the expression of specific chemokines in epithelial cells. Bioinformatics analysis and mutagenesis-based experiments identified Ser413 and Ser424 as key regulatory sites in IRF6. Phosphomimetic mutation of these residues resulted in greatly enhanced IRF6 dimerization and trans-activator function. Collectively, our findings suggest that, in addition to its importance for epithelial barrier function, IRF6 also contributes to host defense by providing specificity to the regulation of inflammatory chemokine expression by TLR2 in epithelial cells.