Gluconobacter oxydans is widely used in several biotechnological applications, where sorbitol or mannitol is commonly used as carbon source at high concentration. In this study, a membrane-bound glucose dehydrogenase-deficient strain (GDHK) was constructed to eliminate growth problems on glucose caused by direct oxidation of glucose in the medium. To achieve improved growth properties for the GDHK strain on glucose, a laboratory adaptive evolution experiment was performed with glucose as the sole carbon source. Results indicated evident, albeit modest, improvements in cell growth after a 50-day (about 430 generations) experimental evolution on glucose. The maximum specific growth rate and biomass yield of the resulting GDHE50 strain were increased around 1.35- to 1.4-fold compared with those of the GDHK strain. Meanwhile, two types of biotransformation reactions using resting cells of G. oxydans were investigated. Significant elevations in biotransformation performance of the GHDE50 strain were observed in comparison with that of the wild-type strain. In addition, resting cells of the GDHE50 strain grown on a relatively low concentration of glucose (10 g/l) could catalyze the biotransformation of glycerol to dihydroxyacetone and ethylene glycol to glycolic acid as efficient as the wild-type G. oxydans cultured on higher concentration of sorbitol or other carbon sources. These results suggest very favorable prospects of using glucose to lower production cost in many important industrial biocatalysis and biotransformation processes.