In present study, a Cecropin‐like peptide from Antheraea pernyi (ApCec) was cloned and characterized. The full‐length ApCec cDNA encoded a protein with 64 amino acids including a putative 22‐amino‐acid signal peptide, a 4‐amino‐acid propeptide, and a 38‐amino‐acid mature peptide. ApCec gene was highly expressed in Malpighian tubules of A. pernyi after induction for 24 h by Escherichia coli in PBS. Pro‐ApCec (including propeptide and mature peptide) and M‐ApCec (just mature peptide) were synthesized chemically and analyzed by HPLC and mass spectroscopy. The antibacterial activity of M‐ApCec is more potent than pro‐ApCec against E. coli K12 or B. subtilus in both minimum inhibitory concentration and inhibition zone assays. Hemolytic assay results showed M‐ApCec possessed a low cytotoxicity to mammalian cells. The secondary structure of M‐ApCec forms α‐helical structure, shown by circular dichroism spectroscopy. Transmission electron microscopy analysis suggested that M‐ApCec killed bacteria by disrupting bacterial cell membrane integrity. Our results indicate ApCec may play an important role in defending from pathogenic bacteria in A. pernyi, and it may be as a potential candidate for applications in antibacterial drug development and agriculture.