As a cytoplasmic parasite, RNA virus develops sophisticated mechanisms to counter host defense and utilize host proteins to facilitate its replication. Here we found Moloney leukemia virus 10 (MOV10), a highly conserved cellular protein belonging to SF1 helicase family, played critical roles in EV71 infection. Silencing cellular MOV10 could restrict EV71 replication, while over-expressing MOV10 resulted in increased viral replication at low dosage and repressed viral replication at high dosage. Further investigation showed that MOV10 exhibited dual functions in EV71 regulation, its C-terminus positively regulated viral replication by binding to EV71 cloverleaf-like structure and the internal ribosome entry site while the N-terminus showed a potential antiviral activity when individually overexpressed. In addition, RNA-dependent interaction between MOV10 and HuR as well as the co-localization of MOV10 and processing bodies were also observed post infection. Taken together, our data indicate a crucial role of MOV10 in EV71 infection for the first time, providing new insights for its roles in EV71 infection. (C) 2016 Elsevier Inc. All rights reserved.