Intracellular nucleic acid sensors detect microbial RNA and DNA and trigger the production of type I interferon. However, the cytosolic nucleic acid-sensing system remains to be fully identified. Here we show that the cytosolic nucleic acid-binding protein LRRFIP1 contributed to the production of interferon-Β (IFN-Β) induced by vesicular stomatitis virus (VSV) and Listeria monocytogenes in macrophages. LRRFIP1 bound exogenous nucleic acids and increased the expression of IFN-Β induced by both double-stranded RNA and double-stranded DNA. LRRFIP1 interacted with Β-catenin and promoted the activation of Β-catenin, which increased IFN-Β expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-Β enhanceosome via IRF3. Therefore, LRRFIP1 and its downstream partner Β-catenin constitute another coactivator pathway for IRF3-mediated production of type I interferon.