Enterovirus A71 (EV-A71) is an important emerging virus posing a threat to children under five years old. Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recognized to play important roles in the onset and development of viral diseases. However, it has not been determined which specific circRNAs are involved in the pathological mechanisms of EV-A71 infection. In this study, RNA-sequencing (RNA-seq) was conducted to characterize differentially expressed circRNAs during the process of EV-A71 infection. Overall, 8726, 10405 and 4710 circRNAs were detected in the control, EV-A71-12 h and EV-A71-24 h groups, respectively, of which 1851 and 951 circRNAs were differentially expressed in the EV-A71-12 h and EV-A71-24 h groups versus the control group. The overlapping circRNAs in the EV-A71-infected groups were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which further demonstrated that the host genes of these circRNAs were principally implicated in activities associated with the progression of viral infection, such as immune system process, Wnt signaling pathway, etc. Additionally, qRT-PCR detection showed that six selected circRNAs were identical to the sequencing data. To excavate the key circRNAs in EV-A71 infection, we comprehensively evaluated and integrated the relationship of circRNA/miRNA/mRNA, and eventually screened 2 key circRNA regulatory axes, namely hsa_circ_0017115/hsa-miR-150-5p/EGR1 and hsa_circ_0005060/hsa-miR-4685-5p/MMP2. In summary, our findings not only provide the first comprehensive expression and functional profile of circRNAs in response to EV-A71 infection, but also offer a novel direction to elucidate the molecular mechanism underlying viral pathogenesis and the cellular immune response in host-EV-A71 interactions.Copyright © 2021. Published by Elsevier B.V.