Multiple gut antimicrobial mechanisms are coordinated in space and time to efficiently fight foodborne pathogens. In Drosophila melanogaster , production of reactive oxygen species (ROS) and antimicrobial peptides (AMPs) together with intestinal cell renewal play a key role in eliminating gut microbes. An alternative protective mechanism would be to selectively prevent the penetration of the intestinal tract by pathogenic bacteria while allowing colonization by commensals. Using real-time imaging to follow the fate of ingested bacteria, we demonstrate that while commensal Lactiplantibacillus plantarum freely enter and cross the larval midgut, pathogenic strains such as. Erwinia carotovora or Bacillus thuringiensis , are actively locked down in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. Supplementing larval food with hCGRP, the human homolog of Dh31, is sufficient to trigger lockdown, suggesting the existence of a conserved mechanism. While the IMD pathway is essential for eliminating the trapped bacteria, it is dispensable for the lockdown. Genetic manipulations impairing bacterial lockdown results in abnormal colonization of posterior midgut regions by pathogenic bacteria. This ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense. Our study reveals a temporal orchestration during which pathogenic bacteria, but not innocuous, are compartimentalized in the anterior part of the midgut in which they are eliminated in an IMD pathway dependent manner.