The current nitrogen fertilization for sugarcane production in Guangxi, the major sugarcane-producing area in China, is very high. We aim to reduce nitrogen fertilization and improve sugarcane production in Guangxi with the help of indigenous sugarcane-associated nitrogen-fixing bacteria. We initially obtained 196 fast-growing bacterial isolates associated with the main sugarcane cultivar ROC22 plants in fields using a nitrogen-deficient minimal medium and screened out 43 nitrogen-fixing isolates. Analysis of 16S rRNA gene sequences revealed that 42 of the 43 nitrogen-fixing isolates were affiliated with the genera Enterobacter and Klebsiella. Most of the nitrogen-fixing enterobacteria possessed two other plant growth-promoting activities of IAA production, siderophore production and phosphate solubilization. Two Enterobacter spp. strains of NN145S and NN143E isolated from rhizosphere soil and surface-sterilized roots, respectively, of the same ROC22 plant were used to inoculate micropropagated sugarcane plantlets. Both strains increased the biomass and nitrogen content of the sugarcane seedlings grown with nitrogen fertilization equivalent to 180 kg urea ha^-1, the recommended nitrogen fertilization for ROC22 cane crops at the seedling stage. ¹⁵N isotope dilution assays demonstrated that biological nitrogen fixation contributed to plant growth promotion. These results suggested that indigenous nitrogen-fixing enterobacteria have the potential to fix N₂ associated with sugarcane plants grown in fields in Guangxi and to improve sugarcane production.