Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea–induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.