Tuber melanosporum, known as the black diamond of cuisine, is highly appreciated for its unique and characteristic aroma, which is mainly due to its volatile organic sulfur-containing compounds (VOSCs). In this work, by adding 5 g/L l-methionine to the fermentation medium, the activities of aminotransferase and alpha-ketoacid decarboxylase were significantly enhanced by 103 and 250 %, respectively, while the activities of alcohol dehydrogenase and demethiolase were decreased by 277 and 39 %. Then, the six VOSCs, i.e., methanethiol (MTL), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 3-(methylthio)propanal (methional), and 3-(methylthio)-1-propanol (methionol), were first detected in the submerged fermentation of T. melanosporum. These results indicated that the biosynthesis of VOSCs was triggered by aminotransferase and alpha-ketoacid decarboxylase. The production of methional and methionol increased with the increased concentrations of l-methionine (i.e., 5, 10, 15, and 20 g/L) before day 4 of the culture protocol, and methionol was the major product in the Ehrlich pathway. The production of MTL was significantly decreased after day 4 with a significantly increased DMDS, and DMDS was the major product of the demethiolation pathway. Compared with the demethiolation pathway with a total flux of sulfur of 11.33-24.32 mu M, the Ehrlich pathway with a total flux of sulfur of 6,149-10,330 mu M was considered the major pathway for the biosynthesis of VOSCs. This is the first report linking the metabolism of l-methionine to the biosynthesis of VOSCs by the Ehrlich and demethiolation pathways during the submerged fermentation of T. melanosporum.