VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3Dpol) and the transfer of UMP by 3Dpol to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3Dpol) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3Dpol by>90%. None of the Site-311 mutations affected the RNA elongation activity of 3Dpol, which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3Dpol, which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3Dpol. Mutation of a polymerase active site in 3Dpol and Site-311 in 3Dpol remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro. These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3Dpol during EV71 VPg uridylylation, such that one 3Dpol presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3Dpol, which in turn catalyzes VPg¡VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.