Antigen presentation by Major Histocompatibility Complex (MHC) and MHC-I like molecules is central to the cellular arm of adaptive immunity.  While the cellular pathways of peptide and lipid antigen presentation by MHC and CD1 molecules are well established, our understanding of the presentation of small molecule metabolites is unclear.  MR1 is an MHC-I like molecule that presents vitamin B metabolites and while diverse cell types ubiquitously express MR1, it only egresses to the cell surface in the presence of Ag(s). We show that, unlike other Ag-presenting molecules, human MR1 does not appear to constitutively present self-ligands, but accumulates in the endoplasmic reticulum (ER) in a ligand-receptive conformation. Here, a lysine residue within MR1 forms a Schiff base with vitamin B metabolites, thereby acting as a “molecular switch” that allows folding and egress of MR1 molecules. Newly formed MR1-antigen complexes follow the secretory pathway, whereupon the half-life of MR1-antigen complexes expressed on the plasma membrane was several hours and not significantly increased when ligand was bound. After exposure on the plasma membrane, MR1-Ag complexes underwent endocytosis into an early/recycling endosomal compartment. While internalized MR1 was measured recycling back to the cell surface and a small degree of ligand exchange could be detected, the most efficient loading of activating ligand occurred on ER-resident MR1. Thus MR1 presentation is characterised by an off-on-off mechanism, where in the steady state the majority of MR1 molecules reside in the ER until they encounter extracellular metabolites. Accordingly the MR1-mediated antigen presentation pathway of vitamin B metabolites possesses features distinct from that of the constitutive presentation of peptide and lipid Ags.