Porcine Parvovirus (PPV) is a pathogen causing porcine reproductive disorders. Non-structural protein NS1 appears diverse functions acting as a predominant regulator in promoting PPV replication. In this study, we identified a PPV NS1 binding protein coatomer subunit epsilon (COP?), and found that COP? is a critical regulator during PPV replication. In NS1 transfected or PPV infected cells, COP? was interacted with NS1 and translocated into nucleus together with NS1. Knockout of COP? could inhibit PPV production by increasing the expression levels of IFN-β, while overexpression of COP? enhanced PPV production by reducing the expression levels of IFN-β. Furthermore, the domain mapping assay showed that the N-terminal amino acids domain of NS1 (25-EAFSYVF-31) were required for the interaction of COP? with NS1. Sequence alignment result displays that parvovirus NS1 (EAFSYVF) amino acids domain is highly conservative among PPV, CPV, FPV and MEV, and down-regulation of COP? could also significantly reduce the replication of these viruses. Notably, we found that the interaction of COP? with NS1 play an important role in promoting the production of type I interferon during PPV or CPV infection, which affect the replication of these viruses. Taken together, the results presented here show a novel function of NS1 interaction with COP? that regulates the parvovirus replication through modulating the type I interferons signaling pathway, provided a potential target for the control of parvovirus-associated diseases.