AbstractActivation of interferon regulatory factor (IRF)‐3 and/or IRF‐7 drives the expression of antiviral genes and the production of α/β IFN, a hallmark of antiviral responses triggered by Toll‐like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF‐3 and/or IRF‐7 but is driven by IRF‐1. IRF‐1 together with myeloid differentiation factor 88 (MyD88) or IL‐1 receptor‐associated kinase (IRAK)‐1 triggered IFN‐β promoter activation. IRF‐1 physically interacted with MyD88 and activation of mDC via TLR‐9 induced IRF‐1‐dependent IFN‐β production paralleled by rapid transcriptional activation of IFN‐stimulated genes. The NF‐κB‐dependent production of pro‐inflammatory cytokines, however, was not influenced by IRF‐1. TLR‐9 signaling through this pathway conferred cellular antiviral resistance while IRF‐1‐deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR‐9 activation of mDC and macrophages contributes to antiviral immunity via IRF‐1.See accompanying commentary: http://dx.doi.org/10.1002/eji.200637009