Mucosa-associated invariant T (MAIT) cells are unconventional T cells recognizing microbial vitamin B2 metabolites presented by the evolutionarily conserved MR1 molecules to mediate innate-like immune protection of human tissues. Here, we analyzed MAIT cells in human organ donor-matched blood, mucosal barrier- and lymphoid tissues and observed a donor-dependent MAIT cell compartment size across tissues with distinct compartmentalization patterns across body sites. Further, paired data revealed tissue-dependent MAIT cell heterogeneity: Intestinal intra-epithelial MAIT cells presented an immunoregulatory CD39high CD27low phenotype. In contrast, a MAIT cell phenotype expressing the adhesion receptor NCAM1 (CD56) was dominant in the liver and indicative of trained effector capacity. CD56+ MAIT cells demonstrated an increased response magnitude and polyfunctionality following multimodal activation. Immuno-secretome analysis highlighted a CD56+ effector signature supporting tissue homeostasis, immune cell recruitment, and inflammation. CD56+ MAIT cells showed a transcriptional profile skewed towards regulation through innate-like pathways or degranulation and exhibited a reduced TCR repertoire diversity. Importantly, CD56 was dynamically upregulated by MAIT cells to a persistent steady state equilibrium following exposure to antigen or homeostatic IL-7 without the need of proliferation. In summary, we demonstrate MAIT cell heterogeneity across human barrier tissue sites and provide evidence for the emergence of a specialized CD56-associated MAIT cell enhanced effector signature.