Abstract: Objective To clone and express the mtlD gene encoding mannitol-1-phosphate dehydrogenase from Vibrio cholerae mannitol specific PTS operon. Methods PCR was conducted to amplify the mtlD gene with the chromosome of N16961 as template. The PCR product was cloned as NdeⅠ-XhoⅠ fragment into the expression vector pET-30a. The mtlD expression was induced with 0.1 mmol/L IPTG, the cell lysis supernatant treated with sonication was purified with Ni-NTA affinity column. The enzyme activity was tested with chromometry assay. Results mtlD was successfully cloned into pET-30a and expressed in the E.coli host cell BL21. The purified MtlD protein containing 6His-tag at its carboxyl terminus showed specific enzyme activity. Conclusion Mannitol-1-phosphate dehydrogenase of V. cholerae was functionally expressed in E. coli as a His-tagged recombinant protein and purified to homogeneity, which severed as the basis for further function study.