As the major causative agent of Hand-Foot-and-Mouth Disease (HFMD) in China, Enterovirus 71 (EV71) has resulted in about 2, 000 deaths since 2008, mostly children aged less than 3 years. EV71, as a member of A species of the genus Enterovirus within the family Picornaviridae, contains a single-stranded positive-sense poly-adenylated RNA genome. The viral replication process is initiated by VPg uridylylation, during which the hydroxyl group of the third tyrosine residue of the virally encoded protein VPg is covalently linked to two UMP molecules by RNA-dependent RNA polymerase (RdRp; also known as 3D protein). We solved the crystal structure of EV71 3D protein complexed with VPg at 2.7 Å resolution by molecular replacement method. Based on uridylation assay and reverse genetics method, we proved that EV71 possesses a novel VPg binding site. Moreover, we proved that EV71 3D protein employs an in vitro trans uridylation mechanism.