Sterile α motif and HD domain-containing protein (SAMHD1) is the only deoxyribonucleosidetriphosphohydrolase in humans to break down dNTPs into the deoxy-bases and triphosphates toregulate dNTP molecular levels in a nucleotide- and metal-dependent mechanism. SAMHD1 playscritical roles in antiviral defense, cancer biology, and cell cycle progression. Therefore, SAMHD1is a crucial therapeutic target for viral restriction and cancer treatment. Human SAMHD1 employsa strict allosteric regulation to oligomerize into active tetramers upon nucleotide binding. Wecarried out bioinformatic analyses that demonstrated the unexpected occurrence of SAMHD1 ingiant viruses (<em>Mimiviridae </em>family), which are comparable to bacteria in size and genomecomplexity but also become infected by phages. Previous studies identified a defense system in<em>Mimivirus </em>to help protect against virophages. Based on this, we hypothesize that viral SAMHD1splay an alternative antiviral strategy, similar to mammals, to deplete dNTP levels and preventvirophage replication. We aim to investigate the diversity and evolutions of SAMHD1 in giantviruses<em> </em>utilizing bioinformatic tools and uncover details of the metallocofactor and allostericactivation of <em>Kv</em> SAMHD1. We found that <em>Mimiviridae</em> SAMHD1 sequences cluster based ongenus, and in the phylogenetic tree, sequences are segregated into two major branches mostlybased on the environmental niches of their hosts. We selected SAMHD1 from <em>Klosneuvirus KNV1</em>(<em>Kv </em>SAMHD1) and <em>Acanthamoeba polyphaga mimivirus</em> (<em>Ap</em> SAMHD1) orthologs for furtherstudy. Like <em>Hs </em>SAMHD1, <em>Kv</em> SAMHD1 harbors a dinuclear metal center at the active site. Themutation of a conserved histidine residue at the active site disrupts the coordination of the secondmetal ion and abolishes the enzymatic activity. We also discovered that allosteric site 1 is essentialfor the allosteric activation of <em>Kv </em>SAMHD1, while allosteric site 2 is not critical for activity butregulates the allosteric inhibition by guanosine-based nucleotides. In summary, <em>Kv</em> SAMHD1shares core mechanistic features with <em>Hs</em> SAMHD1, albeit with less strict allosteric regulation.Our work sheds light on elucidating the activity and allosteric regulation of viral SAMHD1s andproposing that SAMHD1 acts as an auxiliary virophage defense system in this family of viruses.