Simple SummaryAntimicrobial peptides (AMPs) are major components of the insect innate immune system and are involved in multiple antimicrobial and antiviral responses. The black soldier fly is an insect that has received substantial attention in recent years; however, few functional studies on its antimicrobial peptides have been performed. In this study, we specifically induced the expression of the antimicrobial peptide genesHiCG13551andHidiptericin-1of the black soldier fly in the midgut of silkworms.Hidiptericin-1expression in the midgut of silkworms helped to enhance its immune response and imparted superior resistance againstStaphylococcus aureusinfection, whileHiCG13551expression in silkworms tended to weaken the antimicrobial effect. In addition, we performed transcriptome sequencing of midgut tissues afterS. aureusinfection to explore the expression of immune-related genes in the overexpressed strain and found that endogenous antimicrobial peptides, reactive-oxygen-species-related genes, pattern recognition receptors, and immunomodulatory factors were up-regulated in the silkworms due to the transgenic overexpression ofHidiptericin-1. All these results indicated that the ov-AMP49 had better antibacterial activity.AbstractAntimicrobial peptides are molecules with strong antimicrobial activity and are of substantial interest for the immunization of insects. As a type of dipteran insect that can turn organic waste into animal feed, the black soldier fly (BSF) can “turn waste into treasure”. In this study, we investigated the antimicrobial activity of the antimicrobial peptide genes,HiCG13551andHidiptericin-1,of BSF in silkworms, by overexpressing the genes specifically in the midgut. Changes in the mRNA levels of the transgenic silkworms after infection withStaphylococcus aureuswere evaluated using transcriptome sequencing. The results showed thatHidiptericin-1had stronger antimicrobial activity thanHiCG13551. KEGG enrichment analysis showed that the differentially expressed genes in the transgenic overexpressedHidiptericin-1silkworm lines from the D9L strain were mainly enriched in the starch and sucrose metabolism, pantothenate and CoA biosynthesis, drug metabolism (other enzymes), biotin metabolism, platinum drug resistance, galactose metabolism, and pancreatic secretion pathways. In addition, immune-related genes were up-regulated in this transgenic silkworm strain. Our study may provide new insights for future immune studies on insects.