Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.