Abstract We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coliDH5a to stimulate the antibacterial peptide followed by antibacterial activity assay, induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment. The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest, and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation. A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 cells in 16 hours after stimulation. Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus, Escherichia coli K12D31 and Salmonella derby. It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli K12D31 and Salmonella derby.