RIG-I-MAVS signaling pathway plays a crucial role in defending against pathogen infection and maintaining immune balance. Upon detecting viral RNA, RIG-I triggers the formation of prion-like aggregates of the adaptor protein MAVS, which then activates the innate antiviral immune response. However, the mechanisms that regulate the aggregation of MAVS are not yet fully understood. Here, we identified WDR77 as a MAVS-associated protein, which negatively regulates MAVS aggregation. WDR77 binds to MAVS proline-rich region through its WD2-WD3-WD4 domain and inhibits the formation of prion-like filament of recombinant MAVS in vitro. In response to virus infection, WDR77 is recruited to MAVS to prevent the formation of its prion-like aggregates and thus downregulate RIG-I-MAVS signaling in cells. WDR77 deficiency significantly potentiates the induction of antiviral genes upon negative-strand RNA virus infections, and myeloid-specific Wdr77-deficient mice are more resistant to RNA virus infection. Our findings reveal that WDR77 acts as a negative regulator of the RIG-I-MAVS signaling pathway by inhibiting the prion-like aggregation of MAVS to prevent harmful inflammation.