Vitamin K-dependent post-translational modification of glutamate (Glu) residues to γ-carboxyglutamic acid (Gla) by γ-glutamylcarboxylase (GGCX) is essential for blood coagulation, vascular calcification, signalling and cancer cell proliferation in chordate vitamin K-dependent proteins (VKDPs) that contain a Gla domain. By searching in bioinformatic databases using human VKDPs as probes, many novel Gla domain-containing proteins were found in non-chordates. Scrutiny of these revealed that they contained several conserved features in comparison with Gla domain proteins in humans. These included propeptide GGCX recognition residues, cysteine residues and a similar spatial arrangement of glutamate residues. Comparison of the carboxylation enzyme sequences in non-vertebrate and human revealed that most ligand-binding and catalytic residues were conserved. Computational prediction of the subcellular location of these non-vertebrate proteins suggested that most had an extracellular location. Domain prediction revealed they contained diverse domain architectures such as EGF, TyrKc, and FA58C, suggesting that they are likely to function in calcium binding, signalling and cell adhesion. These findings suggest that vitamin K-dependent modification of Gla-domain proteins is conserved in non-vertebrate lineages and originated before the evolution of vertebrates.