D-Glycerate 3-kinase (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C₂ cycle, an indispensable ancillary metabolic pathway to the photosynthetic C₃ cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C₂ cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO₂, providing direct evidence of the obligatory nature of the ultimate step of the C₂ cycle. The newly identified GLYK is both structurally different from and phylogenetically unrelated to known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.