The Ly6 superfamily, present in most metazoan genomes, codes for different cell-surface proteins and secreted ligands containing an extracellular motif called a Ly6 domain or three-finger domain. We report the identification of 36 novel genes coding for proteins of this family in Drosophila. One of these fly Ly6 proteins, coded by the gene boudin (bou), is essential for tracheal morphogenesis in the fly embryo and contributes to the maintenance of the paracellular barrier and the organisation of the septate junctions in this tissue. Bou, a glycosylphosphatidylinositol anchored membrane protein, is also required for septate junction organisation in epithelial tissues and in the chordotonal organ glial cells, but not in the central nervous system. Our study reveals interesting parallelisms between the Ly6 proteins of flies and vertebrates, such as the CD59 antigen. Similarly to this human protein, Bou travels from cell to cell associated with extracellular particles and, consistently, we show that it is required in a non-cell-autonomous fashion. Our work opens the way for future studies addressing the function of Ly6 proteins using Drosophila as a model system.