Dihydroxyacetone (DHA) is an important ketose sugar, which is extensively used in the cosmetic, chemical, and pharmaceutical industries. DHA has been industrially produced by Gluconobacter oxydans with a high demand of oxygen. To improve the production of DHA, the gene vgb encoding Vitreoscilla hemoglobin (VHb) was successfully introduced into G. oxydans, where it was stably maintained, and expressed at about 76.0 nmol/g dry cell weight. Results indicated that the constitutively expressed VHb improved cell growth and DHA production in G. oxydans under different aeration conditions. Especially at low aeration rates, the VHb-expressing strain (VHb(+)) displayed 23.13% more biomass and 37.36% more DHA production than those of VHb-free strain (VHb(-)) after 32 h fermentation in bioreactors. In addition, oxygen uptake rate (OUR) was also increased in VHb(+) strain relative to the control strain during fermentation processes.