Abstract: Objective:To investigate the role of enterovirus type 71 (EV71) 2A protease and host protein ubiquitin-specific protease 4 (USP4) in the infection of EV71. Methods:The expression levels of USP4 mRNA in human rhabdomyosarcoma cells (RD) infected by EV71 were detected by real-time fluorescent quantitative PCR, and the protein levels of VP1 and USP4 were detected by western blot. After knocking down USP4 in host cells, the expression levels of VP1 mRNA and virus titers were detected. The cleavage reason of USP4 protein in host cells was investigated by over-expressing 2A protease. Results:Real-time PCR results showed that the expression levels of USP4 began to decrease at 8 h after EV71 infection (1.03±0.04 and 0.83±0.02 for 0 h and 8 h, respectively, t=4.50, P＜0.05). Western blot results showed that the expression levels of USP4 began to decrease and appeared cleavage bands at 8 h after EV71 infection (1.25±0.01 and 0.51±0.02 for 0 h and 8 h, respectively, t=57.32, P＜0.05). After knocking down USP4 in host cells for 8 hours, the expression levels of VP1 mRNA in the experimental group were significantly higher than that in the control group (3.66±0.08 vs 1.48±0.08, t=17.51, P＜0.05), and the virus titers in the experimental group were also significantly higher than that in the control group \[(10.55±0.29)×10⁵ PFU/mL vs (8.20±0.17)×10⁵ PFU/mL, t=6.98, P＜0.05\]. When 2A protease was over-expressed, the host protein USP4 appeared a cleavage band. Conclusion:USP4 may participate in the antiviral immune response and positively regulate the antiviral signaling pathway. EV71 may escape the immune response and promote the viral replication via splitting host protein USP4 by 2A protease.