RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however， whether RNAi plays antiviral roles in mammals remains unclear. Here， using human enterovirus 71 (HEV71) as a model， we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired， the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO， and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice， whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus， RNAi can function as an antiviral immunity， which is induced and suppressed by a human virus， in mammals.