Abstract Proinsulin-based protease-resistant altered peptide ligands modulate proinsulin-reactive T cells to secret the anti-inflammatory cytokine TGF-β1. Proinsulin is a major diabetes-associated autoantigen. APL have been shown to manipulate the immune response of T cells. Here, we generated a lysosomal protease-resistant proinsulin74–90-derived APL using a CS-directed amino acid modification approach. These prAPL activated TGF-β1 secretion in proinsulin-reactive T cells from PBMC of patients with T1D. We provide evidence that proinsulin-derived prAPL modulate the cytokine signature of proinsulin-reactive T cells at a micromolar range by increasing anti-inflammatory cytokines, including TGF-β1. Thus, the use of prAPL is a promising tool to mitigate autoaggressive T cells.