To better understand Clostridium tetani toxinogenesis, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment shows that the amino acids catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a fermentation map which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene were downregulated at the transcriptional level. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell-adhesion, pyrimidine biosynthesis and salvage, flagellar motility, prophage genes and genes downstream a cobalamin riboswitch. Overall, the fermentation map shows that, mediated by free amino acids concentration, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.