The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDKapm exhibits unique features such as a shorter Kpn-loop, a structural motif previously reported to be part of the active site and involved in oligomer formation. Enzymatic activity measurements on the recombinant NDKapm revealed its preferential affinity for deoxypyrimidine nucleotides. This property might represent an adaptation of NDKapm to the production of the limiting TTP deoxynucleotide required for the replication of the large A+T rich (72%) viral genome. The NDKapm might also assume a role in dUTP detoxification to compensate for the surprising absence of Mimivirus dUTPase (deoxyuridine triphosphate pyrophosphatase) an important enzyme conserved in most viruses. Although the phylogenetic analysis of NDK sequences sampled through organisms from the three domains of life is only partially informative, it favors an ancestral origin for NDKapm over a recent acquisition from a eukaryotic organism by horizontal gene transfer.