Type I IFN production is tightly controlled by host to generate efficient viral clearance without harmful immunopathology or induction of autoimmune disorders. Epigenetic regulation of type I IFN production in innate immunity and inflammatory disorders remains to be fully understood. Several tumor suppressors have been shown to regulate immune response and inflammation. However, the non-classical functions of tumor suppressors in innate immunity and inflammatory diseases need further identification. Here we report retinoblastoma protein (Rb) deficiency selectively enhanced TLR- and virus-triggered production of IFN-β which thus induced more IFN-α generation in the later phase of innate stimuli, but had no effect on the production of TNF, IL-6 and early phase IFN-α in macrophages. Rb1(fl/fl)Lyz2cre(+) Rb-deficient mice exhibited more resistant to lethal virus infection and more effective clearance of influenza virus. Rb selectively bound Ifnb1 enhancer region, but not the promoter of Ifna4, Tnf and Il6, by interacting with c-Jun, the component of IFN-β enhanceosome. Then Rb recruited HDAC1 and HDAC8 to attenuate acetylation of Histone H3/H4 in Ifnb1 promoter, resulting in suppression of Ifnb1 transcription. Therefore, Rb selectively inhibits innate IFN-β production by enhancing deacetylation of Ifnb1 promoter, exhibiting a previous unknown non-classical role in innate immunity, which also suggests a role of Rb in the regulation of type I IFN production in inflammatory or autoimmune diseases.