Interest in MHC-E-restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)-restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.