Contact-dependent growth inhibition (CDI) is a type of competitive mechanisms and has been identified in various strains including Burkholderia, Dickeya, E. coli and Yersinia. Classical CDI systems contain three genes, cdiB, cdiA and cdiI. CdiB encoded by cdiB gene is a conserved β-barrel protein and required for export of CdiA. CdiA protein encoded by cdiA gene includes a conserved N-terminal domain and variable C-terminal toxic domain (CdiA-CT). Immunity protein CdiI binds and inactivates toxin protein CdiA-CT. Here, we identified two CDI systems, an intact cdiBAI operon with a truncated CdiB due to an unexpected mutation and an 'orphan' cdiA-CT/cdiI module in the probiotic Escherichia coli Nissle 1917 (EcN) genome. Both CdiA-CTs from EcN showed auto-inhibition activity when transferring into E. coli DH5α, as well the sequential deletion of amino acid residues resulted in the generation of the most potent mutant of CdiA-CT. CdiI neutralized the toxicity activity of CdiA and was immunity protein as previous report. In conclusion, this is the first report that the functional CDI system is in probiotic EcN and might provide a potential competitive mechanism for probiotic EcN in intestinal microenvironment.