In this paper, we describe the development of VCUSM2, a live metabolic auxotroph of Vibrio cholerae O139. Auxotrophy was achieved by mutating a house keeping gene, hemA, that encodes for glutamyl-tRNA reductase, an important enzyme in the C5 pathway for delta-aminolevulenic acid (ALA) biosynthesis, which renders this strain dependent on exogenous ALA for survival. Experiments using the infant mouse and adult rabbit models show that VCUSM2 is a good colonizer of the small intestine and elicits greater than a four-fold rise in vibriocidal antibodies in vaccinated rabbits. Rabbits vaccinated with VCUSM2 were fully protected against subsequent challenge with 1 x 10(11) CFU of the virulent wild type (WT) strain. Experiments using ligated ileal loops of rabbits show that VCUSM2 is 2.5-fold less toxic at the dose of 1 x 10(6) CFU compared to the WT strain. Shedding of VCUSM2 in rabbits were found to occur for no longer than 4 days and its maximum survival rate in environmental waters is 8 days compared to the greater than 20 days for the WT strain. VCUSM2 is thus a potential vaccine candidate against infection by V. cholerae O139.