DNA viruses typically eject genomic DNA into the nuclei of host cells after entry. It is unclear, however, how nuclear pathogen-derived DNA triggers innate immune responses. We report that heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) recognizes pathogenic DNA and amplifies interferon-alpha/beta (IFN-alpha/beta) production. Upon DNA virus infection, nuclear-localized hnRNPA2B1 senses viral DNA, homodimerizes, and is then demethylated at arginine-226 by the arginine demethylase JMJD6. This results in hnRNPA2B1 translocation to the cytoplasm where it activates the TANK-binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway, leading to IFN-alpha/beta production. Additionally, hnRNPA2B1 facilitates N-6-methyladenosine (m(6)A) modification and nucleocytoplasmic trafficking of CGAS, IFI16, and STING messenger RNAs. This, in turn, amplifies the activation of cytoplasmic TBK1-IRF3 mediated by these factors. Thus, hnRNPA2B1 plays important roles in initiating IFN-alpha/beta production and enhancing stimulator of interferon genes (STING)-dependent cytoplasmic antiviral signaling.