We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+). Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+). Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate. We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).