Cathelicidin antimicrobial peptides (human LL-37 and mouse CRAMP) are mainly virucidal to enveloped virus. However, the effects and relative mechanisms of LL-37 and CRAMP on non-enveloped virus are elusive. We herein found that CRAMP expression was significantly up-regulated post non-enveloped Enterovirus 71 (EV71) infection in different tissues of newborn ICR mice, while EV71 replication gradually declined post CRAMP up-regulation, indicating the antiviral potential of cathelicidin against EV71. In vitro antiviral assay showed that LL-37 and CRAMP markedly reduced cytopathic effects (CPE), intracellular viral RNA copy numbers, viral VP1 protein levels, and extracellular virons in U251 cells post EV71 infection, indicating that LL-37 and CRAMP significantly inhibited EV71 replication. Mechanism of action assay showed that LL-37 and CRAMP were not virucidal to EV71, but markedly regulated antiviral immune response in U251 cells. Co-incubation of LL-37 or CRAMP with U251 cells markedly increased the basal interferon-β (IFN-β) expression and interferon regulatory transcription factor 3 (IRF3) phosphorylation, modestly enhanced IFN-β production and IRF3 phosphorylation upon EV71 infection, and significantly reduced interleukin-6 (IL-6) production and p38 mitogen-activated protein kinase (MAPK) activation post EV71 infection. Additionally, LL-37 and CRAMP directly inhibited viral binding to U251 cells. Collectively, LL-37 and CRAMP markedly inhibited EV71 replication via regulating antiviral response and inhibiting viral binding, providing potent candidates for peptide drug development against EV71 infection.