The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are major sensors against viral infection, but their roles in DNA virus infection largely remain unknown. This study found that a previously uncharacterised protein, pS183L, negatively regulates RLR signalling by suppressing MDA5 oligomerisation. Specifically, we showed that the overexpression of pS183L suppresses MDA5 but not cGAS-STING or RIG-I-induced IFN-β activation. Consistently, pS183L inhibited high molecular weight poly (I:C) activated IFN-β production. Furthermore, we demonstrated that pS183L interacts with CARDs and the MDA5 Helicase domain, consequently blocking MDA5 oligomerisation and the MDA5-MAVS interaction. Taken together, we concluded that pS183L blocks MDA5 oligomerisation through protein-protein interaction and thus disrupts MDA5-mediated IFN-β signalling.