MAIT cells recognize microbial metabolites derived from the riboflavin biosynthesis pathway. This pathway is constantly active in aerotolerant bacteria residing in the colon mucosa, resulting in the steady-state synthesis of 5-OP/E-RU in the gut. Surprisingly, 5-OP-RU can cross epithelia and travel to the thymus, where it can be captured by various cell types and contribute to MAIT cell thymic maturation. Key questions remain regarding the intriguing relationship between MAIT cells and the microbiota. In particular, the mechanisms of 5-OP-RU passage and transport in the body, and the potential effect of chronic low-grade TCR stimulation in the absence of infection, remain unclear. Here, we found that 5-OP-RU administered intra-rectally becomes available in blood within 1 minute and remains in circulation for about 6 hours. 5-OP-RU remains free in serum and is not stabilized by proteins. In vitro experiments on epithelial cell monolayers suggest a passive, bi-directional diffusion most likely through tight junctions. MAIT cells from all tested organs including liver, lung, thymus, gingiva and meninges receive TCR signals upon intra-rectal administration of 5-OP-RU, implying the presence of MR1-expressing cells at steady-state across tissues. 5-OP-RU administered intra-rectally induces transient MAIT cell responses marked by the expression of survival and cell-cycle genes, suggesting that MAIT cells may accumulate in response to chronic TCR triggering by microbiota-derived ligands. Accordingly, MAIT cell frequencies increase with age up to 30-fold in adult conventionally-housed mice, but not in germ-free mice. MAIT cells adoptively transferred into Mr1-/- recipients fail to maintain in non-lymphoid tissues, and MAIT cell frequencies gradually decline upon Mr1 deletion in adult mice. Mono-colonization with Escherichia coli, which produces 5-OP-RU but not the recently described ligand CA7S, is sufficient to induce MAIT cell accumulation overtime. Thus, commensal bacteria constantly release riboflavin pathway-derived ligands that passively cross the gut barrier and provide low-grade stimulation to MAIT cells, driving their accumulation overtime.