Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has broken out several times and was accompanied by neurological disease. microRNAs, a class of small non-coding RNAs that are approximately 20 nucleotides long, play important roles in the regulation of various biological processes, including antiviral defense. However, the roles of miRNAs in EV71 replication and pathogenesis are not well understood. In this study, we found that the expression of miR-27a was significantly decreased in EV71-infected cells. Interestingly, the over-expression of miR-27a could inhibit EV71 replication, as measured by virus titration, qPCR, and Western blotting. We identified EGFR mRNA is a bona fide target of miR-27a by computational analysis and luciferase reporter assays. Furthermore, miR-27a could decrease EGFR expression, as measured by qPCR and Western blotting. Moreover, the inhibition of EGFR expression by miR-27a decreased the phosphorylation of Akt and ERK, which facilitate EV71 replication. These results suggest that miR-27a may have antiviral activity against EV71 by inhibiting EGFR.