In myeloid dendritic cells, activation of the IL-27p28 gene is selectively induced by ligands of TLR4 or TLR3, both coupled to the Toll/IL-1R-related domain-containing adaptor-inducing IFN/IFN regulatory factor (IRF)3 pathway. In response to both ligands, autocrine type 1 IFN production was required for optimal IL-27p28 expression. Type I IFN signaling was necessary for sustained IRF1 activation and formation of the IRF9-containing IFN-stimulated gene factor 3 complex. Indeed, we demonstrated that IRF1 and IRF9 are sequentially activated and recruited to the IL-27p28 IFN-stimulated regulatory element site. Involvement of IRF1 and IRF9 in the induction of IL-27p28 was confirmed in vitro and upon in vivo exposure to TLR ligands. Thus, in response to TLR4 or TLR3 ligation, the initial induction of the IL-27p28 gene depends on the recruitment of IRF1 and IRF3, whereas transcriptional amplification requires recruitment of the IFN-stimulated gene factor 3 complex. These results highlight the complex molecular interplay between TLRs and type I IFNs for the control of IL-27 synthesis.