Enterovirus 71 (EV71) is a picornavirus that can cause severe neurological complications in children. Like other picornaviruses, the genomic RNA of EV71 contains a long 5′ untranslated region (UTR). Cellular proteins interact with the EV71 5′ UTR, and these interactions are important for virus replication. Using an RNA pull-down assay and proteomics approaches, this study identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) as one of the EV71 5′ UTR-associated proteins. The interaction between hnRNP K and the 5′ UTR was further confirmed by mapping the interaction regions to stem–loops I–II and IV in the 5′ UTR. During EV71 infection, hnRNP K was enriched in the cytoplasm where virus replication occurs, whereas hnRNP K was localized in the nucleus in mock-infected cells. Viral yields were found to be significantly lower in hnRNP K knockdown cells and viral RNA synthesis was delayed in hnRNP K knockdown cells in comparison with negative-control cells treated with small interfering RNA. These results suggest that hnRNP K interacts with the EV71 5′ UTR and participates in virus replication.