Major histocompatibility-complex (MHC) class I-related (MR1) protein presents vitamin B-related metabolites to Mucosal-Associated Invariant T (MAIT) cells. Microbial riboflavin precursors are well known MR1 ligands; however, it remains unclear whether host-generated riboflavin degradation metabolites influence MR1-mediated immunity. Here, we report that riboflavin catabolites, including 10-formylmethylflavin (FMF), lumichrome, lumiflavin and alloxazine bind to MR1 with moderate affinity, while riboflavin itself binds weakly. While the microbial riboflavin ligands are known to increase MR1 cell surface presentation, the riboflavin catabolites were found to moderately reduce cell surface levels of MR1 by stabilizing and retaining MR1 in the endoplasmic reticulum (ER). Our data showed that these riboflavin catabolites bind to the intracellular MR1 and inhibit MR1 release from the ER. They also weakly competed with vitamin B-based antigens (Ags) for MR1 binding, thereby inhibited MAIT cell activation. The crystal structures of MR1 complexed with riboflavin, FMF, lumichrome and lumiflavin, show binding of these three-ringed compounds in the A'-pocket of MR1. The crystal structure of MR1-lumichrome revealed that lumichrome formed a covalent “flavin bond” with MR1-Lys43 differing from the typical Schiff-base bond of MR1-Lys43-Ag complexes seen with riboflavin precursors. Collectively, we identified three-ringed isoalloxazine derivatives which can bind MR1 and downregulate cell surface expression levels, suggesting a potential role in dampening MAIT cell immunity.