BackgroundChemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol (2,3-BDO) is a promising biological plant growth promoter.ResultsIn this study, we attempted to develop an effective strategy for the biological production of highly pureR,R-2,3-butanediol (R,R-2,3-BDO) byPaenibacillus polymyxafermentation. First, gamma-ray mutagenesis was performed to obtainP. polymyxaMDBDO, a strain that grew faster than the parent strain and had high production ofR,R-2,3-BDO. The activities ofR,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity ofR,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L ofR,R-2,3-BDO with high optical purity (> 99% of C4products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L ofR,R-2,3-BDO at 60 h. The fed-batch fermentation broth ofP. polymyxaMDBDO, which contained highly pureR,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings.ConclusionsThis study suggests thatP. polymyxaMDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purityR,R-2,3-BDO can be produced at high concentrations usingP. polymyxa.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-023-02133-y.