UNLABELLED: Foot-and-mouth disease virus (FMDV), a member of the IMPORTANCE: Viral RNA replication is the key stage in understanding the pathogenic mechanisms of foot-and-mouth disease virus (FMDV). During this process, the viral non-structural protein 3D serves as an RNA-dependent RNA polymerase (RdRp) to synthesize progeny RNA using the viral genomic RNA as a template. However, the regulatory effect of host cells on FMDV 3D proteins has not yet been studied. In this study, we find that heat shock protein A1 (HSPA1) degrades the viral 3D protein through the chaperone-mediated autophagy (CMA) pathway, thereby inhibiting the RNA replication of FMDV and interfering with virus infection. This study, for the first time, demonstrates that HSPA1 employs its chaperone function to mediate the degradation of the FMDV RdRp.