MHC class I-related molecule (MR1) is an evolutionary conserved antigen presenting molecule. MR1 presents bacteria-derived metabolites to mucosal associated invariant T (MAIT) cells. However, host-derived ligands of MR1 are not fully understood. In this study, we searched for novel ligand(s) of MR1 from mouse tissues using column chromatography-based separation and mass spectrometry together with a sensitive reporter assay. We purified an active component distinct from known antigens and determined the chemical structure as sulfated bile acid. This compound is a host-derived metabolite mainly present in the liver and gut, but is also detected in the thymus. When we established mice lacking the biosynthesis pathway of this metabolite, the development of thymic MAIT cells was severely impaired, whereas it was rescued by the administration of the synthetic bile acid sulfate. In human peripheral MAIT cells, bile acid sulfate induced cell survival but not proliferation, in sharp contrast to 5-OP-RU which promotes MAIT cell proliferation. Furthermore, the gene ontology that characterized the bile metabolite stimulation included wound healing and homeostatic immune regulation while the bacterial antigen induced pro-inflammatory genes. These results suggest that a host-derived metabolite controls the development and homeostasis of MAIT cells.