During replication, numerous viral RNAs are modified by N-6-methyladenosine (m(6)A), the most abundant internal RNA modification. m(6)A is believed to regulate elements of RNA metabolism, such as splicing, stability, translation, secondary structure formation, and viral replication. In this study, we assessed the occurrence of m(6)A modification of the EV71 genome in human cells and revealed a preferred, conserved modification site across diverse viral strains. A single m(6)A modification at the 5' UTR-VP4 junction was shown to perform a protranslational function. Depletion of the METTL3 methyltransferase or treatment with 3-deazaadenosine significantly reduced EV71 replication. Specifically, METTL3 colocalized with the viral dsRNA replication intermediate in the cytoplasm during EV71 infection. As a nuclear resident protein, METTL3 relies on the binding of the nuclear import protein karyopherin to its nuclear localization signal (NLS) for nuclear translocation. We observed that EV71 2A and METTL3 share nuclear import proteins. The results of this study revealed an inner mechanism by which EV71 2A regulates the subcellular location of METTL3 to amplify its own gene expression, providing an increased understanding of RNA epitranscriptomics during the EV71 replication cycle. (C) 2020 Published by Elsevier Inc.