The main effectors in innate immune system of B. mori L. are antimicrobial peptides (AMPs). We imunizied B. mori with varied inoculum size of Pseudomonas aeruginosa ATCC 25668 cells to relate the importance of inoculum size towards AMP production. There is a sharp change in λmax from 278 nm to 285 nm (bathochromic shift) in the hemolymph of immunized silkworm incubated for 24 h. FTIR study of the hemolymph extracted from the immunized B. mori showed peak at 1550 cm-1 indicates the presence of α-helical peptides in the hemolymph.The peptide fraction was obtained through methanol, acetic acid and water mixture (90:1:9) extraction, followed by peptide purification using RP-HPLC. The fraction exhibiting antibacterial property was collected and characterized further using MALDI-TOF mass spectrometry. A linear -helical peptide with flexible termini (LLKELWTKMKGAGKAVLGKIKGLL -Bm-Frac6), corresponding to a previously described peptide from ant venom and denominated ponericin-L1. This peptide showed antibacterial activity against P. aeruginosa. Moreover, MTT assay and hemolytic study revealed that the Bm-Frac6 (B. mori ponericin-L1) is non-toxic toward mammalian healthy cells. Finally, our study shows that silkworm larvae challenged with P. aeruginosa also induced modification in physiological processes, leading to significant morpho-anatomical changes.