Acetoin, a major extracellular catabolic product of Bacillus subtilis cultured on glucose, is widely used to add flavor to food and also serves as a precursor for chemical synthesis. The biosynthesis of acetoin from pyruvate requires the enzymes α-acetolactate synthase (ALS) and α-acetolactate decarboxylase (ALDC), both of which are encoded by the alsSD operon. The transcriptional regulator ALsR is essential for the expression of alsSD. Here we focused on enhancing the production of acetoin by B. subtilis using different promoters to express ALsR. The expression of reporter genes was much higher under the control of the HpaII promoter than under control of the P bdhA promoter. Although the HpaII promoter highly enhanced transcription of the alsSD operon through overexpression of ALsR, the production of acetoin was not significantly increased. In contrast, moderate enhancement of ALsR expression using the P bdhA promoter significantly improved acetoin production. Compared with the wild-type, the enzyme activities of ALS and ALDC in B. subtilis harboring P bdhA were increased by approximately twofold, and the molar yield of acetoin from glucose was improved by 62.9 % in shake flask fermentation. In a 5-L fermentor, the engineered B. subtilis ultimately yielded 41.5 g/L of acetoin. Based on these results, we conclude that enhanced expression of ALDC and ALS by moderately elevated expression of the transcriptional regulator ALsR could increase acetoin production in recombinant B. subtilis.