The signal peptide region of preproinsulin (PPI) contains epitopes targeted by human leucocyte antigen-A (HLA-A)-restricted (HLA-A0201, A2402) cytotoxic T-cells as part of the pathogenesis of β-cell destruction in type 1 diabetes. We extended PPI epitope discovery to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles revealing that 4/6 alleles present epitopes derived from the signal peptide region. During co-translational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical, proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter-associated-with-antigen-processing (TAP)-dependent, and ER-luminal (TAP-independent) epitopes, each presented by different HLA class I molecules, and N-terminal trimmed by ER aminopeptidase 1 (ERAP1) for optimal presentation. In vivo, TAP expression is significantly up-regulated and correlated with HLA class I hyper-expression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.