Mucosal-associated invariant T (MAIT) cells are a key mediator of microbe-immune communication. These abundant unconventional T cells recognise derivatives of microbial riboflavin biosynthesis via their semi-invariant T cell receptor and perform diverse roles in antimicrobial defence and barrier homeostasis. In mice, the presence of the major MAIT antigen 5-OP-RU in early life is both necessary and sufficient for complete MAIT cell development. In humans, circulating MAIT cell abundance and phenotype varies widely between individuals, but the drivers of this variation are unknown. By combining microbiome and immune profiling of diverse human populations from the UK, Zambia, Kenya and Uganda with microbial culture and functional assays, we demonstrated that the gut microbiome is a significant driver of MAIT cell abundance, activation phenotype and functionality. Notably, we identified riboflavin metabolism by methanogenic archaea as a key driver of 5-OP-RU availability and MAIT cell phenotype. Archaea are a distinct domain of life to bacteria and their role in the gut microbiome remains poorly understood. Archaeal cultures, including the human commensal Methanobrevibacter smithii, produced MAIT cell antigens in vitro and activated MAIT cells via a TCR-dependent mechanism. We therefore show for the first time that MAIT cells can sense the gut archaeome, demonstrating a novel role for archaea in shaping human immunity.