MAIT cells are abundant innate-like T cells with a highly conserved TCR recognizing ligands derived from the microbial riboflavin biosynthetic pathway (5-OP/OE-RU) or from the bile acid metabolism (CA7S). MAIT cells accumulate during childhood to reach high frequencies in adults, but the underlying mechanisms are unclear. The intestinal microbiota constantly produces MAIT ligands which contribute to MAIT cell thymic maturation early in life. How MAIT ligands cross the intestinal barrier and reach distant tissues is unknown. The impact of chronic low-grade TCR stimulation on MAIT cells is also unclear. We found that 5-OP-RU administered intra-rectally becomes available in blood within less than a minute indicating rapid passage across intestinal barrier. Preliminary in vitro experiments with colonocyte-goblet cell monolayers suggest a passive transport through tight junctions. After passage, 5-OP-RU remains in circulation for about 6 hours consistent with its half-life in aqueous solutions in vitro. Blood partitioning indicates that 5-OP-RU remains free in serum and is not stabilized by proteins. Intra-rectal administration induces Nur77 expression in MAIT cells from all tissues including liver, spleen, lung but also thymus and meninges, supporting that 5-OP-RU gets transported to all organs and indicating the presence of 5-OP-RU-presenting cells across the body at steady state. Sterile 5-OP-RU stimulation triggers a transient transcriptional response associated with low-level proliferation, raising the possibility that MAIT cells accumulate in response to chronic TCR triggering by microbiota-derived ligands. Accordingly, MAIT cell frequencies increase with time in B6 and B6-MAITCast mice housed in pathogen-free conditions, but not in germ-free mice. Mono-colonization with Escherichia coli, which produces 5-OP/E-RU but not CA7S, is sufficient to induce MAIT cell accumulation overtime. Thus, intestinal bacteria constantly release riboflavin pathway-derived ligands that passively cross epithelial barriers and provide low-grade stimulation to MAIT cells, possibly contributing to their accumulation overtime.