Background. Enterovirus 71 (EV71) can cause severe diseases and even lead to death in children. There is no vaccine or specific antiviral therapy to prevent or cure EV71 infection. Although interferon (IFN)-α has been used in the treatment of several viral infections, we found that IFN-α alone was ineffective in restricting EV71 replication in Vero cells. Methods. Through a bioinformatics analysis, several cellular proteins in the IFN response pathway were identified as susceptible substrates that might be degraded by the EV71-encoded 3C protease (3Cpro). Results. Indeed, IRF9 was shown to be vulnerable to 3Cpro cleavage, as revealed by enzyme-based and cell-based assays. Thus, the IFN-mediated antiviral mechanism compromised by the viral 3Cpro in EV71-infected cells may be accountable, at least partially, for that IFN-α cannot inhibit EV71 replication. Because rupintrivir (AG7088) is known to be an effective EV71 inhibitor, we investigated the effects of the combination of rupintrivir and IFN-α on EV71 replication and found that they strongly synergized with each other in inhibiting EV71 replication. Conclusions. Because rupintrivir was shown to be generally tolerable in earlier clinical investigations, it is worth evaluating whether a combination of rupintrivir and IFN-α could be an effective treatment for EV71.