Hydrogenobacter thermophilus TK-6 is a thermophilic, hydrogen-oxidizing bacterium that fixes carbon dioxide as a sole carbon source via the reductive tricarboxylic acid cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes in the pathway. Strain TK-6 has at least two isozymes of OGOR, namely For and Kor. These OGORs showed different expression patterns under aerobic conditions than under anaerobic conditions. In this work, we developed a homologous recombination method for Hydrogenobacter, and constructed a For mutant and a Kor mutant. Observation of phenotypes of the mutant strains showed that Kor was essential for anaerobic growth and that For activity supported robust aerobic growth of the organism.