Enterovirus 71 (EV71) causes major outbreaks of hand, foot, and mouth disease. Host factors and signaling pathways exhibit important functions in the EV71 life cycle. We conducted algorithm analysis based on miRNA profiles and their target genes to identify the miRNAs and downstream signaling pathways involved in EV71 infection. The miRNA profiles of human rhabdomyosarcoma cells treated with interferon (IFN-)-α or IFN-γ were compared with those of cells infected with EV71. Genes targeted by differentially expressed miRNAs were identified and assigned to different signaling pathways according to public databases. The results showed that host miRNAs specifically responded to the viral infection and IFN treatment. Some miRNAs, including miR-124 and miR-491-3p, were regulated in opposite manners by the IFNs and EV71. Some signaling pathways regulated by both EV71 infection and IFN treatment were also predicted. These pathways included axon guidance, Wingless/Int1 (Wnt) signaling cascade, platelet-derived growth factor receptor (PDGFR)/PDGF, phosphatidylinositol 3-kinase (PI3K), Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK), transforming growth factor-beta receptor (TGF-βR)/TGF-β, SMAD2/3, insulin/insulin-like growth factor (IGF), bone morphogenetic protein (BMP), CDC42, ERB1, hepatocyte growth factor receptor (c-Met), eukaryotic translation initiation factor 4E (eIF4E), protein kinase A (PKA), and IFN-γ pathways. The identified miRNA and downstream signaling pathways would help to elucidate the interaction between the virus and the host. The genomics method using algorithm analysis also provided a new way to investigate the host factors and signaling pathways critical for viral replication.