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Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication., Author Summary Many plus-strand RNA viruses encode a viral RNA polymerase and capping enzymes to synthesize a 5’-capped RNA genome. However, how these two activities are coordinated during viral replication is not understood. In flaviviruses, polymerase and capping enzymes are encoded in a single multifunctional protein, where separate domains within the polypeptide are responsible for these activities; flavivirus NS5, composed of the polymerase and methyltransferase domains, carries out viral RNA synthesis, 5’-RNA capping, and RNA cap methylations. Previous NS5 monomer structures were unable to provide mechanistic insight into how the two domains communicate or the quaternary organization of the functional enzyme. We have determined the crystal structure of dengue virus NS5 and show that the NS5 dimer is likely the biological assembly of NS5, and RNA synthesis and RNA capping may be coordinated by the dimer. We found that essential interactions between the two NS5 domains can be maintained either within a monomer or via inter-molecular interactions within a dimer, and thus NS5 can adopt multiple conformations while preserving necessary interactions between the methyltransferase and polymerase domains. Using dengue virus, we additionally determined that such specific interaction between the two NS5 domains is the major determinant of viral replication.