Abstract: (R,R)-2,3-butanediol is an important four-carbon platform compound with a variety of applications in liquid crystal materials, high value added chiral compounds, especially in asymmetric synthesis of optical active pharmaceuticals. α-acetolactate synthase gene alsS, α-acetolactate decarboxylase gene alsD and（R,R）-2,3-butanediol dehydrogenase gene R,R-bdh from Paenibacillus polymyxa DSM365 were connected to the expression vector pMA5. The main metabolic pathway of（R,R）-2,3-butanediol was enhanced by introducing P. polymyxa DSM-365 to construct an engineering P. polymyxa strain DM-5 which can efficiently synthesize（R,R）-2,3-butanediol. The production of（R,R）-2,3-butanediol via DM-5 was up to 54.91 g/L in fed-batch fermentation, with a yield of 0.52 g/g and a productivity of 0.92 g·L-1·h-1 after 60 h. Comparing with the parent strain, the yield of（R,R）-2,3-butanediol increased by 19.66%, and the concentration of ethanol and acetoin decreased, methanol with little change. The results of this study showed that the overexpression of key genes alsS, alsD and R,R-bdh in P. polymyxa could significantly improve the yield and production intensity of（R,R）-2,3-butanediol, providing reference for metabolic engineering and industrial production of（R,R）-2,3-butanediol in P. polymyxa.