The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30 min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-α expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.