ABSTRACTEnterovirus D68 (EV-D68) is a globally emerging pathogen causing severe respiratory illnesses mainly in children. The protease from EV-D68 could impair type I interferon (IFN-I) production. However, the role of the EV-D68 structural protein in antagonizing host antiviral responses remains largely unknown. We showed that the EV-D68 structural protein VP3 interacted with IFN regulatory factor 7 (IRF7), and this interaction suppressed the phosphorylation and nuclear translocation of IRF7 and then repressed the transcription of IFN. Furthermore, VP3 inhibited the TNF receptor associated factor 6 (TRAF6)-induced ubiquitination of IRF7 by competitive interaction with IRF7. IRF7Δ305–503showed much weaker interaction ability to VP3, and VP3Δ41–50performed weaker interaction ability with IRF7. The VP3 from enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) was also found to interact with the IRF7 protein. These results indicate that the enterovirus structural protein VP3 plays a pivotal role in subverting host innate immune responses and may be a potential target for antiviral drug research.IMPORTANCEEV-D68 is a globally emerging pathogen that causes severe respiratory illnesses. Here, we report that EV-D68 inhibits innate immune responses by targeting IRF7. Further investigations revealed that the structural protein VP3 inhibited the TRAF6-induced ubiquitination of IRF7 by competitive interaction with IRF7. These results indicate that the control of IRF7 by VP3 may be a mechanism by which EV-D68 represses IFN-I production.