Compared with traditional pathogenic producers, Bacillus subtilis as a Class I microorganism offers many advantages for industrial-scale 2,3-butanediol production. Unlike previous reports in which two stereoisomers (with a ratio of 3:2) were produced, we first found that wild type B. subtilis 168 generates only D-(-)-2,3-butanediol (purity >99%) under low oxygen conditions. The total high yield of 2,3-butanediol and acetoin, and acetoin reductase enzyme assay indicate that it is the high level of NADH availability, instead of high acetoin reductase activity, contributes more to 2,3-butanediol production in B. subtilis. The strategy for increasing the pool of NADH availability, the key factor for 2,3-butanediol production, was designed through low dissolved oxygen control, adding reducing substrates and rationally metabolic engineering. A transhydrogenase encoded by udhA was introduced to provide more NADH from NADPH and allowed enhanced 2,3-butanediol production. Finally, BSF20 produced 49.29 g/L D(-)- 2,3-butanediol. These results demonstrated that B. subtilis is a competitive producer for chiral 2,3-butanediol production. (C) 2014 Wiley Periodicals, Inc.