Regulatory T cells (Treg) maintain immune homeostasis due to their anti-inflammatory functions. They can be generated either centrally in the thymus or in peripheral organs. Metabolites such as short chain fatty acids produced by intestinal microbiota can induce peripheral Treg differentiation, by activating G-protein-coupled-receptors like GPR109A. In this study, we identified a novel role for GPR109A on thymic Treg development. We found that Gpr109a-/- mice had increased Treg under basal conditions in multiple organs compared to wild type mice (WT). GPR109A was not expressed on T cells but on medullary thymic epithelial cells (mTECs), as revealed by single cell RNA sequencing in both mice and humans and confirmed by flow cytometry in mice. mTECs isolated from Gpr109a-/- mice had higher expression of autoimmune regulator (AIRE), the key regulator of Treg development, while the subset of mTECs that did not express Gpr109a displayed increased Aire expression and enhanced signalling related to mTEC functionality as well. Increased thymic Treg in Gpr109a-/- mice was associated with protection from experimental autoimmune encephalomyelitis, with ameliorated clinical signs and reduced inflammation. This work identifies a novel role for GPR109A and by extension, the gut microbiota, on thymic Treg development via its regulation of mTECs.