L-Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d-amino acids (d-aa) have important non-ribosome-based functions 1. Mammals synthesize d-Ser and d-Asp, primarily in the central nervous system, where d-Ser is critical for neurotransmission 2. Bacteria synthesize a largely distinct set of d-aa, which become integral components of the cell wall and are also released as free d-aa 3, 4. However, the impact of free microbial d-aa on host physiology at the host–microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d-aa that are derived from the microbiota. Furthermore, the microbiota induces production of d-amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d-aa by DAO, which yields the antimicrobial product H 2 O 2, protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d-aa and DAO as previously unrecognized mediators of microbe–host interplay and homeostasis on the epithelial surface of the small intestine.