Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease in which the insulin-producing beta cells are destroyed. Post-translational modifications, such as the conversion of glutamine (Q) residues to glutamic acid (E) by deamidation, have been proposed to enhance the immunogenicity of self-antigens. This is best illustrated in coeliac disease where enzymatic deamidation of gliadin increases its reactivity to autoreactive CD4+ T-cells. The inflammatory conditions associated with T1D can lead to activation of the deamidating enzyme tissue transglutaminase 2 (tTG2). Full-length C-peptide is a major autoantigen in T1D that harbours many CD4+ T-cell epitopes and has four glutamines that are potential sites for tTG2-mediated deamidation. We hypothesized that deamidation of full-length C-peptide would increase its immunogenicity to CD4+ T-cells. To investigate this, Jurkat cells were transduced with T-cell receptors (TCRs) from C-peptide specific CD4+ T-cell clones isolated from the pancreatic islets, or peripheral blood, of donors with T1D. These different Jurkat lines were tested for reactivity against a panel of C-peptide variants with single glutamine to glutamic acid substitutions and one with all four glutamines substituted with glutamic acid. The epitopes of all Jurkat lines were known. For 13 of the 18 (72%) Jurkat lines tested, recognition was destroyed when a glutamine was replaced by glutamic acid within the epitope known to be recognized by TCR. To assess responses against deamidated and unmodified C-peptide in the peripheral blood of people with or without T1D we used the CFSE-based proliferation assay. Although increased responses to at least one deamidated peptide, compared to native C-peptide, were observed from all T1D samples, no deamidated peptide was consistently more or less stimulatory than native C-peptide. Collectively we find no evidence that the deamidation of C-peptide increases its immunogenicity in type 1 diabetes.