Retinoic acid-inducible gene I-like receptor (RLR) is one of the most important pattern recognition receptors of the innate immune system that detects positive and/or negative stranded RNA viruses. Subsequently, it stimulates downstream transcription of interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) inducing the production of interferons (IFNs) and inflammatory cytokines. Tumour necrosis factor receptor associated factor 6 (TRAF6) is a key protein involved in the RLR-mediated antiviral signalling pathway, recruiting additional proteins to form a multiprotein complex capable of activating the NF-κB inflammatory pathway. Despite TRAF6 playing an important role in regulating host immunity and viral infection, the deubiquitination of TRAF6 induced by viral infection remains elusive. In this study, we found that enterovirus 71 (EV71) infection attenuated the expression of Ubiquitin-specific protease 4 (USP4) in vitro and in vivo, while overexpression of USP4 significantly suppressed EV71 replication. Furthermore, it was found that EV71 infection reduced the RLR signalling pathway and enhanced the degradation of TRAF6. USP4 was also found to interact with TRAF6 and positively regulate the RLR-induced NF-κB signalling pathway, inhibiting the replication of EV71. Therefore, as a novel positive regulator of TRAF6, USP4 plays an essential role in EV71 infection by deubiquitinating K48-linked ubiquitin chains.