Biomass accumulation is a key factor for successful biosynthesis of chemicals in industrial scale. As an important platform for biorefinery, Klebsiella pneumoniae has been widely used for the production of various chemicals. However, low production of chemicals is often ascribed to insufficient biomass. Herein we demonstrated that activation of dha regulon can efficiently promote biomass accumulation of K. pneumoniae in both minimal and nutrient medium. Transcriptome analysis revealed that dha regulon was activated by supply of 5 g/L glycerol in glucose medium, and neither glycerol was consumed nor 1,3-propanediol was produced before glucose was exhausted. Moreover, biofilm formation was retarded but cell motility was accelerated. Interestingly, the susceptibility of K. pneumoniae to common antibiotics was not elevated. Next, mutation and overexpression of the genes in dha regulon were conducted and results demonstrated that loss of the regulator dhaR led to growth defect. On the contrary, overexpression of dhaD, dhaB, dhaR and dhaT contributed partly to the promoted biomass accumulation in glucose medium. In summary, activation of dha regulon of K. pneumoniae enhanced biomass accumulation but had little impact on antibiotic susceptibility. This study provided a promising method for improving the productivity of valuable chemicals in industrial scale.