A naturally cholera toxin gene negative Vibrio cholerae (O1, El Tor, Ogawa) strain, named IEM101, was isolated in China. The human volunteer tests showed that this strain was safe, able to colonize the intestinal mucosa, and able to induce a strong immune response. Also other studies indicated that it was an efficient live vector to deliver heterologous antigens. In this article, a thymidylate synthase gene (thyA)-defined mutant was constructed using homologous recombination. Except for the morphological changes in minimal medium and slightly reduced colonization capacity, mutant strain IEM101-T maintained most of the desirable features as the wild-type strain IEM101 in terms of growth rate and immunogenicity. However, the mutant was more biosafe than its parent strain. In conclusion, IEM101-T may be a promising strain to develop live vaccine candidate of cholera or an attractive vaccine vector to deliver heterologous antigens in vivo.