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Enterovirus 71 (EV71) is the primary pathogen of hand-foot-and-mouth disease (HFMD) in children and virus infections are associated with severe neurological dysfunctions and even death. MIR2911 is a honeysuckle-encoded atypical microRNA with extreme stability. Here, we report that MIR2911 directly inhibits EV71 replication by targeting the VP1 gene. Bioinformatics prediction and luciferase reporter assay showed that MIR2911 could target the VP1 gene of EV71. Transfection experiments using synthetic MIR2911 and extracted RNA from HS decoction shown that each of these preparations was capable of inhibiting EV71 VP1 protein expression; however, these preparations did not impact EV71 mutants in which the MIR2911-binding sites were mutated. Furthermore, EV71 replication was increased by antagomirs against MIR2911 in the HS decoction, implying that MIR2911 was physiologically functional in controlling EV71 replication in vitro. These results indicated that, by targeting VP1 gene, MIR2911 may effectively inhibit EV71 replication. Our results also provide a potential novel strategy on the therapy and/or prevention of HFMD originating from EV71 virus infection.We analyse the anti-EV71 potential of the MIR2911.It cleaves the EV71 genome and binds to the VP1 protein.The inhibitor interferes with viral translation and replication in cell culture.