DExD/H-box helicase 9 (DHX9), or RNA helicase A (RHA), is an abundant multifunctional nuclear protein. Although it was previously reported to act as a cytosolic DNA sensor in plasmacytoid dendritic cells (pDCs), the role and molecular mechanisms of action of DHX9 in cells that are not pDCs during DNA virus infection are not clear. Here, a macrophagespecific knockout and a fibroblast-specific knockdown of DHX9 impaired antiviral innate immunity against DNA viruses, leading to increased virus replication. DHX9 enhanced NF-kappa B-mediated transactivation in the nucleus, which required its ATPasedependent helicase (ATPase/helicase) domain, but not the cytosolic DNA-sensing domain. In addition, DNA virus infection did not induce cytoplasmic translocation of nuclear DHX9 in macrophages and fibroblasts. Nuclear DHX9 was associated with a multiprotein complex including both NF-kappa B p65 and RNA polymerase II (RNAPII) in chromatin containing NF-kappa B-binding sites. DHX9 was essential for the recruitment of RNAPII rather than NF-kappa B p65, to the corresponding promoters; this function also required its ATPase/helicase activity. Taken together, our results show a critical role of nuclear DHX9 (as a transcription coactivator) in the stimulation of NF-kappa B-mediated innate immunity against DNA virus infection, independently of DHX9's DNA-sensing function.