Foot-and-mouth disease virus (FMDV) poses a significant global threat to livestock production, underscoring the urgent need for a deeper understanding of virus-host interactions to develop effective prevention strategies. In this study, we identify prolyl endopeptidase (PREP) as a critical host factor in FMDV infection. We demonstrate that PREP knockout (PREP-KO) significantly enhances host resistance to FMDV infection by upregulating the expression of RIG-I and MDA5 mRNA. Specifically, PREP-KO destabilizes the PUM1 protein via proteasomal and apoptotic degradation pathways, thereby increasing RIG-I promoter activity. Additionally, PREP-KO inhibits the FMDV life cycle at the replication stage. In vivo experiments further reveal that PREP inhibition or knockout provides protection against FMDV infection following a sublethal challenge in mice. Our findings identify PREP as a novel antiviral target, providing valuable insights for development of antiviral therapies and breeding of disease-resistant livestock.