The monomorphic MHC-I-related protein 1 (MR1) presents vitamin metabolites to mucosal-associated invariant T (MAIT) cells, an innate-like subset of T lymphocytes. The best characterised MAIT-activating MR1 ligands are unstable pyrimidine intermediates of riboflavin synthesis, a pathway specific to certain fungi and bacteria and, thus, intrinsically non-self for humans. While it has been reported that soluble, extracellular ligands encounter MR1 in an incompletely folded, ligand-receptive state in the endoplasmic reticulum (ER), it remains to be determined how the antigenic metabolites initially gain access to the cytosol and, ultimately, reach the ER. Here, we use a functional genetic screening technique based on insertional mutagenesis of the near-haploid human cell line HAP1 to discover novel players in MR1 antigen presentation and trafficking. A HAP1 clone overexpressing human MR1 was transduced with a gene trap virus to inactivate genes in an unbiased manner. Subsequently, the mutagenised population was incubated with the MR1-stabilising ligand Acetyl-6-Formylpterin, stained for MR1 surface expression, and analysed by flow cytometry. The tails of the distribution were FACS sorted to enrich for cells in which positive or negative regulators of MR1 were inactivated. Mapping of the viral insertion sites by Illumina deep sequencing allowed identification of genes statistically overrepresented in either of the two sorted populations which constitute putative modulators of MR1 intracellular trafficking or MR1 stability. Among the most significant positive regulators of MR1 surface expression identified in our screen was β₂-microglobulin, binding of which is a known requirement for the surface translocation of MR1. A number of putative regulators of MR1 trafficking were validated using a CRISPR/Cas9 approach and tested in functional and biochemical assays.