Spatially and temporally controlled expression of inflammatory mediators is critical for an appropriate immune response. In this study, we define the role for interferon regulatory factor 5 (IRF5) in secretion of tumor necrosis factor (TNF) by human dendritic cells (DCs). We demonstrate that DCs but not macrophages have high levels of IRF5 protein, and that IRF5 is responsible for the late-phase expression of TNF, which is absent in macrophages. Sustained TNF secretion is essential for robust T-cell activation by DCs. Systematic bioinformatic and biochemical analyses of the TNF gene locus map 2 sites of IRF5 recruitment: 5' upstream and 3' downstream of the TNF gene. Remarkably, while IRF5 can directly bind to DNA in the upstream region, its recruitment to the downstream region depends on the protein-protein interactions with NF-kappaB RelA. This study provides new insights into diverse molecular mechanisms employed by IRF5 to regulate gene expression and implicates RelA-IRF5 interactions as a putative target for cell-specific modulation of TNF expression.