Beta interferon (IFN-beta) gene expression in response to virus infection relies on the dynamic assembly of a multiprotein enhanceosome complex that is initiated by the activation of two inducible transcription factors, interferon regulatory factor 3 (IRF3) and NF-kappaB. Virus or double-stranded RNA-induced activation of IFN-beta gene expression is prevented by the addition of protein deacetylase inhibitors. The isolated IRF-responsive positive regulatory domain was found to require deacetylation for its activity, but IRF3 protein activation leading to its nuclear translocation and DNA binding was not impaired by deacetylase inhibition. In contrast, NF-kappaB activity was not affected by deacetylase inhibitors. RNA interference indicated that several deacetylase enzymes, including histone deacetylase 1 (HDAC1), HDAC8, and HDAC6, influence IFN-beta gene expression with opposing effects. While HDAC1 and HDAC8 repress IFN-beta expression, HDAC6 acts as a coactivator essential for enhancer activity. Virus replication is enhanced in HDAC6-depleted cells, demonstrating HDAC6 is an essential component of innate antiviral immunity.