Enterovirus 71 (EV71) is one of the causative pathogens of hand, foot and mouth disease (HFMD), especially the form associated with fatal neurological disorders. Sustained outbreaks of EV71 infections remain a serious health threat worldwide. However, no antiviral agent against EV71 for clinical therapy has been approved. Retro-2cycl and Retro-2.1 are inhibitors of several pathogens specifically targeting the intracellular vesicle transport, which also participates in the EV71 lifecycle processes including progeny virus release. Here, we reported that Retro-2cycl and Retro-2.1, respectively, could inhibit EV71 infection with 50% effective concentrations of 12.56 μM and 0.05 μM in a cytopathic effect inhibition assay and showed relatively low cytotoxicity with 50% cytotoxicity concentrations of more than 500 μM and 267.80 μM. Preliminary mechanism studies revealed that Retro-2cycl and Retro-2.1 did not inhibit EV71 protein synthesis or RNA replication but could block progeny EV71 release specifically. Furthermore, administration of Retro-2cycl at the dose of 10 mg/kg significantly protected 90% of newborn mice from lethal EV71 challenge. Consequently, our results for the first time identified Retro-2cycl and Retro-2.1 as effective inhibitors of EV71 as well as lead compounds, which would contribute to anti-EV71 drug development. We also identified progeny virus release and the intracellular vesicle transport as antiviral targets for EV71.