Natto as a fermented soybean product has many health benefits for human due to its rich nutritional and functional components. However， the unpleasant odor of natto， caused by the formation of branched-chain short fatty acids (BCFAs)， prohibits the wide acceptance of natto products. This work is to identify the key gene of BCFAs formation and develop the guidance to reduce natto odor. Transcriptional analysis of BCFAs synthesis pathway genes was conducted in two Bacillus subtilis strains with obvious different BCFAs synthesis abilities. The transcriptional levels of bcd， bkdAA， and ptb in B. subtilis H-9 were 2.7-fold， 0.7-fold， and 8.9-fold higher than that of B. subtilis H-4， respectively. Therefore， the ptb gene with the highest transcriptional change was considered as the key gene in BCFAs synthesis. The ptb encoded enzyme Ptb was further characterized by inducible expression in Escherichia coli. The recombinant Ptb protein (about 32 kDa) was verified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The catalysis functions of Ptb were confirmed on substrates of isovaleryl-CoA and isobutyryl-CoA， and the higher catalysis efficiency of Ptb on isovaleryl-CoA explained the higher level of isovaleric acid in natto. The optimal activities of Ptb were observed at 50 °C and pH 8.0， and the enzymatic activity was inhibited by Ca， Zn， Ba， Mn， Cu， SDS， and EDTA. Collectively， this study reports a key gene responsible for BCFAs formation in natto fermentation and provides potential strategies to solve the odor problem.