Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72 mol/mol, a 16.4 % increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66 % in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087 g/l h, using M9 medium plus 30 g glucose/l under micro-aerobic conditions, to 0.273 g/h l using LB medium plus 50 g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20 g/l.