Diarrhea is the most common infection in children under the age of five worldwide. In spite of this, only a few vaccines to treat infectious diarrhea exist, and many of the available vaccines are sparingly and sporadically administered. Major obstacles to the development and widespread implementation of vaccination include the ease and cost of production, distribution, and delivery. Here we present a novel, customizable and self-assembling vaccine platform that exploits theVibrio choleraebacterial biofilm matrix for antigen presentation. We use this technology to create a proof-of-concept, live-attenuated whole cell vaccine that is boosted by spontaneous association of a secreted protein antigen with the cell surface. Sublingual administration of this live-attenuated vaccine to mice confers protection againstV. choleraechallenge and elicits production of antigen-specific stool IgA. The platform presented here enables development of antigen-boosted vaccines that are simple to produce and deliver, addressing many of the obstacles to vaccination against diarrheal diseases. This may also serve as a paradigm for the development of broadly protective, biofilm-based vaccines against other mucosal infections.ImportanceDiarrheal disease is the most common infection afflicting children worldwide. In resource-poor settings, these infections are correlated with cognitive delay, stunted growth, and premature death. With the development of efficacious, affordable, and easily administered vaccines, such infections could be prevented. While a major focus of biofilm research has been their elimination, here we harness the bacterial biofilm to create a customizable platform for cost-effective, whole cell mucosal vaccines that self-incorporate secreted protein antigens. We use this platform to develop a sublingually administered, live-attenuated prototype vaccine based onVibrio cholerae.This serves not only as a proof-of-concept for a multivalent vaccine against common bacterial enteric pathogens but also as a paradigm for vaccines utilizing other bacterial biofilms to target mucosal infections. Copyright 2018 American Society for Microbiology.