The link between mitochondria and the immune system is centered on the energy-producing function of mitochondria in immune cells. Whether mitochondria affect antigen-presentation and the generation of metabolites that stimulate T cells remains unclear. We have found that mitochondrial activity influences the immune response to MR1 via two mechanisms. The first promotes MR1 cell-surface expression and is mediated by complex III and dihydroorotate dehydrogenase enzymatic activity, both required for uridine-dependent compound production. A second mechanism is the generation of uridine-related, thymidine-related, and reactive-oxygen-species-dependent antigens that bind MR1 and stimulate MR1-restricted T cells. One immunogenic compound identified is 5-formyl-deoxyuridine (5-FdU). Structural studies show that 5-FdU binds in the A’-antigen-binding pocket of MR1, with the deoxyribose oriented toward the surface of MR1 available for TCR interaction. This indicates that mitochondria may be involved in the host's response to metabolic alterations, promoting the activation of MR1-restricted T cells.