ยฉ 2018 American Society for Microbiology. Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao People's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing a considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon beta (IFN-ฮฒ) production as well as the activation of transcription factors IRF3 and NF-ฮบB. Interestingly, NS6 does not impede the IFN-ฮฒ promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKKฮต, and IRF3. Further analyses revealed that NS6 is not an RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-ฮฒ production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-ฮฒ production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response.