Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.