Virus infection triggers interferon (IFN)-mediated innate immune defenses in part through viral nucleic acid interactions. However, the immune recognition mechanisms by which the host identifies incoming DNA viruses are still elusive. Here, we show that increased levels of Kaposi's sarcoma-associated herpesvirus (KSHV) persistency are observed in retinoic acid-inducible gene I (RIG-I)-deficient cells and that KSHV ORF64, a tegument protein with deubiqutinase (DUB) activity, suppresses RIG-I-mediated IFN signaling by reducing the ubiquitination of RIG-I, crucial for its activation. This study suggests that RIG-I plays a potential role in sensing KSHV infection and that KSHV ORF64 DUB counteracts RIG-I signaling.