Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from increment dhaK increment derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain increment glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain increment derK increment glpK and the triple deletion strain increment dhaK increment derK increment glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although Delta derK Delta glpK consumed a small amount of DHA in the late phase of growth, increment dhaK Delta derK Delta glpK did not show DHA consumption on glucose-glycerol medium. The transformants of the increment dhaK Delta derK Delta glpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus.