Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells, found within the islets of Langerhans in the pancreas, are destroyed by islet-infiltrating T cells. Identifying the antigenic targets of beta-cell reactive T cells is critical to gain insight into the pathogenesis of T1D and develop antigen-specific immunotherapies. Several lines of evidence indicate that insulin is an important target of T cells in T1D. Because many human islet-infiltrating CD4 T cells recognize C-peptide-derived epitopes, we hypothesized that full-length C-peptide (PI), the peptide excised from proinsulin as it is converted to insulin, is a target of CD4 T cells in people with T1D. CD4 T cell responses to full-length C-peptide were detected in the blood of: 14 of 23 (>60%) people with recent-onset T1D, 2 of 15 (>13%) people with long-standing T1D, and 1 of 13 (<8%) HLA-matched people without T1D. C-peptide-specific CD4 T cell clones, isolated from six people with T1D, recognized epitopes from the entire 31 amino acids of C-peptide. Eighty-six percent (19 of 22) of the C-peptide-specific clones were restricted by HLA-DQ8, HLA-DQ2, HLA-DQ8, or HLA-DQ2, HLA alleles strongly associated with risk of T1D. We also found that full-length C-peptide was a much more potent agonist of some CD4 T cell clones than an 18mer peptide encompassing the cognate epitope. Collectively, our findings indicate that proinsulin C-peptide is a key target of autoreactive CD4 T cells in T1D. Hence, full-length C-peptide is a promising candidate for antigen-specific immunotherapy in T1D.