The fermentation rates of mannitol in toxigenic and non-toxigenic El Tor strains of Vibrio cholerae are obviously different, which is a valuable indicator in the rapid identification of toxigenic strain. To determine the regulating role of mtlR in transcription of mannitol PTS operon in V. cholerae, and whether it plays a role in the ferment difference of the toxigenic and non-toxigenic strains, the mtlR deletion mutants from the mannitol rapid-ferment strain (non-toxigenic strain) and slow-ferment strain (toxigenic strain) were constructed. Comparisons of the growth in M9 containing 0.2% mannitol as the sole carbon source and pH change in mannitol fermentation media of these wild strains and their mutants, indicated that mtlR is a repressor. Its repression in mtlCBA transcription was further verified with the analyses of quantitative reverse-transcriptional PCR. However, the regulation of mtlR is not the immediate cause of the ferment difference of the toxigenic and non-toxigenic strains. The study also provides the necessary data in the analyses of mannitol ferment difference between the toxigenic and non-toxigenic V. cholerae strains.