The nonobese diabetic (NOD) mouse is a good model for human type 1 diabetes, which is characterized by autoreactive T-cell-mediated destruction of insulin-producing islet β-cells of the pancreas. The 9–23 amino acid region of the insulin B-chain [B_(9–23)] is an immunodominant T-cell target antigen in the NOD mouse that plays a critical role in the disease process. By testing a series of B_(9–23) peptide analogs with single or double alanine substitutions, we identified a set of altered peptide ligands (APLs) capable of inhibiting B_(9–23)-induced proliferative responses of NOD pathogenic T-cell clones. These APLs were unable to induce proliferation of these clones. However, vaccinations with the APLs induced strong cellular responses, as measured by in vitro lymphocyte proliferation and Th2 cytokine production (i.e., interleukin [IL]-4 and IL-10, but not γ-interferon [IFN-γ]). These responses were cross-reactive with the native antigen, B_(9–23), suggesting that the APL-induced Th2 responses may provide protection by controlling endogenous B_(9–23)-specific Th1 (i.e., IFN-γ-producing) pathogenic responses. One of these APLs that contained alanine substitutions at residues 16 and 19 (16Y→A, 19C→A; NBI-6024) was further characterized for its therapeutic activity because it consistently induced T-cell responses (e.g., T-cell lines and clones) that were of the Th2 type and that were cross-reactive with B_(9–23). Subcutaneous injections of NBI-6024 to NOD mice administered either before or after the onset of disease substantially delayed the onset and reduced the incidence of diabetes. This study is the first to report therapeutic activity of an APL derived from an islet β-cell-specific antigen in type 1 diabetes.