Mitochondrial single-stranded DNA-binding protein 1 (SSBP1), a component of the mitochondrial DNA replisome, is involved in DNA replication, repair and maintenance of mitochondrial DNA. However, its function in the bovine ephemeral fever virus (BEFV) infection has not been described. Herein, we found that SSBP1 acted as an essential negative regulator of mitochondrial antiviral signaling protein (MAVS) to maintain immune homeostasis. Under BEFV infection, SSBP1 was upregulated in host cells, which boosted the susceptibility to virus. SSBP1 dramatically impaired BEFV-triggered antiviral immune response by degrading MAVS. Mechanistically, SSBP1 induced K48-linked ubiquitination of MAVS catalyzed by Smad ubiquitin regulatory factor 1 (Smurf1), thereby promoting ubiquitinated-MAVS for proteasomal degradation. Most importantly, we identified A01, a Smurf1-specific inhibitor that blocked degradation of MAVS induced by SSBP1 and enhanced MAVS-mediated antiviral signaling, consequently suppressing BEFV replication. Taken together, our findings reveal that SSBP1 restricts BEFV-induced innate immune activation through SSBP1-Smurf1-MAVS signaling axis, indicating its potential role as a therapeutic target for viral infections.