Abstract Introduction: We investigated whether the cholera toxin B subunit (CTB)-specific secretory IgA (SIgA) induced by rice-based oral vaccine expressing CTB (MucoRice-CTB) gives long-term protection against V. cholerae - and enterotoxigenic Escherichia coli (ETEC)-induced diarrhea. Methods: Mice were orally immunized with MucoRice-CTB, which stored at room temperature for 3 years, and then evaluated for induction of SIgA-mediated protective immunity against CT or heat-labile enterotoxin (LT). The role of CTB-specific SIgA was also examined in polyimmunoglobulin receptor (pIgR)-deficient mice. Long-term immune memory was evaluated 6 months after final primary immunization. In vivo V. cholerae (El Tor O1 Inaba) and ETEC challenge was performed in the intestinal loops of immunized mice. Results: Oral MucoRice-CTB immunization provided SIgA-mediated protection against LT- or CT-induced diarrhea. This protection was impaired in pIgR-deficient mice. The vaccine offered long-lasting protection against toxin-induced diarrhea (for at least 6 months after primary immunization), and a single booster immunization extended the length of protective immunity for at least 4 more months. In the intestinal loop assay, MucoRice-CTB vaccination inhibited diarrhea in the face of V. cholerae and ETEC challenge. Conclusion: MucoRice-CTB is an effective cold-chain-free oral vaccine inducing CTB-specific SIgA-mediated long-term protection against V. cholerae - or ETEC-induced diarrhea.