ABSTRACTSmall proteins perform a diverse array of functions, from microbial competition, to endocrine signaling, to building biomaterials. Microbial systems that can produce recombinant small proteins enable discovery of new effectors, exploration of sequence activity relationships, and have the potential forin vivodelivery. However, we lack simple systems for controlling small-protein secretion from Gram-negative bacteria. Microcins are small-protein antibiotics secreted by Gram-negative bacteria that inhibit the growth of neighboring microbes. They are exported from the cytosol to the environment in a one-step process through a specific class of type I secretion systems (T1SSs). However, relatively little is known about substrate requirements for small proteins exported through microcin T1SSs. Here, we investigate the prototypic microcin V T1SS fromEscherichia coliand show that it can export a remarkably wide range of natural and synthetic small proteins. We demonstrate that secretion is largely independent of the cargo protein’s chemical properties and appears to be constrained only by protein length. We show that a varied range of bioactive sequences, including an antibacterial protein, a microbial signaling factor, a protease inhibitor, and a human hormone, can all be secreted and elicit their intended biological effect. Secretion through this system is not limited toE. coli, and we demonstrate its function in additional Gram-negative species that can inhabit the gastrointestinal tract. Our findings uncover the highly promiscuous nature of small-protein export through the microcin V T1SS, which has implications for native-cargo capacity and the use of this system in Gram-negative bacteria for small-protein research and delivery.IMPORTANCEType I secretion systems for microcin export in Gram-negative bacteria transport small antibacterial proteins from the cytoplasm to the extracellular environment in a single step. In nature, each secretion system is generally paired with a specific small protein. We know little about the export capacity of these transporters and how cargo sequence influences secretion. Here, we investigate the microcin V type I system. Remarkably, our studies show that this system can export small proteins of diverse sequence composition and is only limited by protein length. Furthermore, we demonstrate that a wide range of bioactive small proteins can be secreted and that this system can be used in Gram-negative species that colonize the gastrointestinal tract. These findings expand our understanding of secretion through type I systems and their potential uses in a variety of small-protein applications.