Glycine oxidase, encoded by the thiO gene, participates in the biosynthesis of thiamin by providing glyoxyl imine to form the thiazole moiety of thiamin. We have purified and characterized ThiO from Pseudomonas putida KT2440. It has a monomeric structure that is distinct from the homotetrameric ThiOs from Bacillus subtilis and Geobacillus kaustophilus. The P. putida ThiO is unique in that glycine is its preferred substrate, which differs markedly from the B. subtilis and G. kaustophilus enzymes that use D-proline as the preferred substrate.