Expression of genes for Bacillus anthracis toxin and capsule virulence factors are dependent upon the AtxA transcription factor. The mechanism by which AtxA regulates the transcription of its target genes is unknown. Here we report that bioinformatic analyses suggested the presence in AtxA of two PTS (phosphenolpyruvate: sugar phosphotransferase system) regulation domains (PRD) generally regulated by phosphorylation/dephosphorylation at conserved histidine residues. By means of amino acid substitutions that mimic the phosphorylated (H to D) or the unphosphorylated (H to A) state of the protein, we showed that phosphorylation of H199 of PRD1 is likely to be necessary for AtxA activation while phosphorylation of H379 in PRD2 is inhibitory to toxin gene transcription. In vivo labelling experiments with radioactive phosphate allowed us to propose that H199 and H379 are AtxA residues subject to regulated phosphorylation. In support to these notions, we also show that deletion of ptsHI, encoding the HPr intermediate and the EI enzymes of PTS, or growth in the presence of glucose affect positively and negatively, respectively, the activity of AtxA. Our results link virulence factor production in B. anthracis to carbohydrate metabolism and, for the first time, provide a mechanistic explanation for AtxA transcriptional activity.