The paper aims to determine the minimum functional unit of twin-arginine translocation (Tat) system by analyzing the Tat system gene cluster composition, which was deduced by homology comparison between Tat gene cluster of Escherichia coli (E. coli) K12 and Vibrio cholerae (V. cholerae) N16961. Tat mutant strains were constructed by deleting tat genes of the N16961 reference strain either individually or double genes combined together. Tat transport system minimum functional unit were then analyzed by comparing the phenotypes among Tat mutant strain, complement strain and wild strain. The data showed that the Tat system gene cluster of V. cholerae N16961 consisted of tatA, tatB, tatC and tatA₂ , among which, tatA, tatB and tatC located in the chromosomeⅠand tatA₂ gene located in the chromosome Ⅱ. The tatB and tatC genes were indispensable for Tat system functions, while tatA₂ might play a role similar to tatE in E. coli. The tatB and tatC combined together constitutes the minimum functional unit of V. cholera Tat system. TatA and TatA₂ have similar functions and may share the same functional role in the Tat system.