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Abstract: Objective To construct engineering peptide pheromonicin-Clostridium tetani (Ph-CT), and to test its bactericidal activity. Methods We amplified the gene of variable regions from hybridoma cells which secreted monoclonal antibody (mAb) against antigen in the membrane of Clostridium tetani and linked the small antibody mimetic to the channel-forming domain of colicin Ia to create Ph-CT. The Ph-CT was purified by CM sepharose ion-exchange column. Its in vitro antibacterial activity was evaluated by colony culture with different doses of Ph-CT (final concentration 2, 4, 8, and 16 μg/mL，respectively). Then we inoculated culture medium with CT strains and different doses of Ph-CT (final concentration of 4 and 16 μg/mL). The in vitro antibacterial activity of Ph-CT was evaluated by cumulative survival of mice. After 16 hours’ anaerobic culture, the mice was treated with filtered CT medium or CT medium. Results We constructed Ph-CT successfully. CT colonies appeared in the CT medium treated with Ph-CT (2, 4 μg/mL), while no colony appeared in the CT medium treated with Ph-CT (8, 16 μg/mL). All mice survived when they were injected with filtered CT medium treated with Ph-CT (4, 16 μg/mL) and CT medium treated with Ph-CT (16 μg/mL). Three (50%) mice survived when they were injected with CT medium treated with Ph-CT (4 μg/mL). All mice in the control groups died after CT infections. Conclusion Ph-CT may be of value as antibiotics against Clostridium tetani