<p>Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus belonging to the deltacoronavirus genus, has caused massive losses to the livestock industry and poses a potential risk of cross-species transmission. Interferon-stimulated gene 15 (ISG15) plays a crucial role in virus infection and immune regulation through interactions with cellular and viral proteins. However, the mechanisms underlying the effect of ISG15 on PDCoV infection are poorly understood. In this study, we demonstrated that the knockout of ISG15, mediated by CRISPR-Cas9, facilitates PDCoV replication and that ISG15 modification (ISGylation) deficiency attenuates the antiviral ability of ISG15. To clarify the novel roles of ISG15 during PDCoV infection, we constructed a stable ISG15-overexpressed IPEC-J2 cell line. We employed tandem affinity purification followed by mass spectrometry analyses with ISG15, enabling the identification of 538 known and novel candidate ISGylation proteins including proliferating cell nuclear antigen (PCNA) which is a sign of DNA damage. We showed that ISGylation of PCNA significantly inhibited PDCoV replication. During PDCoV infection, HERC5, an ISG15 E3 ligase, interacted with PCNA and promoted its ISGylation, whereas USP18 eventually de-ISGylated PCNA. Our results not only reveal that ISGylation acts as a post-translational modification of PCNA but also elucidate the function of ISGylation in PCNA during PDCoV infection, thereby adding a novel layer of complexity to the virus–host interaction interface.</p>