Human MAIT cells in circulation are remarkably homogenous with regard to phenotype and function, with the exception of a few functional differences associated with the expression of certain surface markers. Some findings have indicated that mucosal tissue localization influences the functional phenotype of MAIT cells. To study this in more detail, we studied MAIT cells isolated from organ donor-matched tissue samples to determine MAIT cell characteristics across individuals and throughout human body sites. Our findings indicate that the MAIT cell compartment size is donor-dependent across tissues with distinct tissue compartmentalization patterns. We identified consistent patterns of tissue adaptation in the tissue resident MAIT cell pool, with a regulatory MAIT cell population dominant in the intestines, as well as an intrahepatic effector signature with enhanced innate characteristics and high responsiveness. Thus, we demonstrate MAIT cell functional heterogeneity across human barrier tissue sites at homeostasis and provide evidence for tissue site-dependent enrichment of regulatory and effector signatures. These findings provide a spatial dimension to our understanding of the MAIT cell compartment and a roadmap to investigate tissue-dependent functions in this conserved part of host immune defense.