Vitamin K epoxide reductase subunit 1 (VKORC1) found within the vitamin K cycle is required in the activation of vitamin K dependent proteins in the vertebrate coagulation pathway and the formation of the bone matrix. Structural studies on the human, rat, and mouse VKORC1 enzymes using mutagenesis and X-ray crystallography have highlighted important domains and residues related to inhibitor and substrate binding. A second paralogous vertebrate enzyme, VKORC1L1, with possible housekeeping functions in protection against antioxidant stress has been established. Both paralogues in human and rat can catalyse the conversion of vitamin K epoxide (VKE) to vitamin K quinone and hydroquinone yet display differences in their inhibition by vitamin K antagonists (VKAs). Presently, the substrate specificity or VKA inhibition of VKORC1 in other vertebrates is not known, nor is there any published data of inhibitor effects. Additionally, the properties and function of VKORs in non-vertebrates that do not have blood or bone is not known. This research expanded on the published phylogeny of VKORs, scrutinising parts of the metazoan VKOR tree yet to be analysed and encompassing greater than 15-fold more VKOR sequences than any other previous comparative study. Additionally, genomic and sequence analysis lead to the conclusion that the two vertebrate VKOR paralogues potentially emerged from VKORC1L1. This investigation also aimed to compare the kinetics and inhibition of the recombinant human and rat VKORC1 with VKORs in other metazoan species. This revealed a gradation of VKA resistance for the studied species, which was consistent with their placement on the Tree of Life. Non-mammalian vertebrates, lower chordates and non-vertebrate VKORs were less sensitive to VKA than their mammalian VKORC1 counterparts. Additionally in vivo experiments with Drosophila melanogaster revealed that inhibition of VKOR led to an increased susceptibility to survival with oxidative stressors suggesting a role in shielding against oxidative stress. In conclusion, this study contributes novel insights into the evolution and functional roles of VKOR enzymes in metazoans, their varying responses to anticoagulants, and potential antioxidant functions.