Mucosa-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have TH1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MAIT cells are restricted by the MHC-related protein (MR1), a highly conserved non-classical Class I molecule. Vitamin B metabolites were identified as ligands that bind and stabilize MR1, allowing for the activation of MAIT cells. The mechanism by which MR1 is loaded with these ligands leading to MAIT cell activation is not understood, and is likely critical to understanding the immunobiology of MAIT cell function. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein at basal conditions or following incubation with ligand. MR1 localized to vesicles that have features of late endosomes. Addition of the ligand 6-formyl pterin resulted in the translocation of MR1 from this vesicular compartment to the cell surface. Given the endosomal location of MR1, we postulated that vesicular trafficking pathways would be integral to the regulation of MR1 ligand loading and surface translocation. As a result, we screened a lentiviral shRNA library specific for genes encoding vesicular trafficking proteins to determine the genes required for presentation of Mtb antigens to an MR1-restricted T cell clone. Knockdown of Sec22b and Syntaxin18 (Stx18) in antigen presenting cells was specifically associated with a decrease in the Mtb-dependent IFN-g release by MAIT cells, but not those restricted by HLA-B45 or HLA-E. Stx18 but not Sec22b knockdown resulted in decreased translocation of ligand-bound MR1 to the cell surface. We hypothesize that Stx18 is required for proper trafficking of MR1 to the cell surface upon loading with ligand, while Sec22b may be required for the interaction of mycobacterial antigens with MR1.