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 nucleotidyl transferase 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’-PO4 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 (Tyr39, Tyr40, Asp48, Asp52) or abolished (Tyr51) sealing of a 5’ PO4 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+.