In this study,an acetoin-producing bacterial strain SF4-3 was isolated from Japanese traditional food natto.When it was incubated with 180 r/min for 96 h in the flask at 37 ℃,acetoin concentration of strain SF4-3 could reach 31.26 g/L using glucose as the main carbon source.The fermentation broths of strain SF4-3 were analyzed by gas chromatography(GC) and gas chromatography-mass spectrometry(GC-MS),the main product was acetoin and its purity was higher than 91%,without butanedione and 2,3-butanediol produced which were commonly present as by-products.Based on morphology,physiological and biochemical characteristics as well as the 16S rDNA gene sequence,strain SF4-3 was identified as B.subtilis.