The introduction of thetoxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in mostVibrio choleraestrains. SuchV. choleraestrains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in theseV. choleraestrains is secreted into the culture medium. In this study,V. choleraestrains that can express intracellular CTB under the control of thetoxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking thectxABpromoter toctxBwithoutctxAand confirmed CTB expression from the plasmid inV. choleraecontaining thetoxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids—from the 7thto the 20thamino acid—of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructedV. choleraestrains in which chromosomalctxABis replaced byntrctxBorntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.