Abstract The antimicrobial peptide diptericin plays an important role in defence against microorganisms. Drosophila melanogaster diptericin mRNA levels showed an increase during the late final instar larval stage when the ecdysteroid titres increase to initiate metamorphosis. Deletion analysis in Drosophila melanogaster mbn2 (mbn2) cells identified a cis ‐regulatory element (AAGAAAGATCCCCTG) necessary for 20‐hydroxyecdysone enhancement of peptidoglycan‐induced expression of diptericin in the 3 kb diptericin promoter. Proteins extracted from mbn2 cells treated with peptidoglycan plus 20‐hydroxyecdysone specifically bound to this element. 20‐hydroxyecdysone also enhanced peptidoglycan‐induced expression of four other antimicrobial peptide (AMP) genes – drosomycin , attacin‐A , metchnikowin and cecropin A1 . Moreover, in silico promoter analysis using the meme program showed that an eight‐nucleotide region of the identified cis ‐regulatory element is present in the promoters of these four AMP genes. These studies suggest that 20‐hydroxyecdysone regulates the expression of AMP genes through a conserved cis ‐regulatory element.