Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation <i>in vivo</i> Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.<b>IMPORTANCE</b> Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide.