A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V. cholerae, The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium. The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo. Overexpression of B-G33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains.