In type 1 diabetes (T1D), autoimmune destruction of insulin-producing b-cells and critically diminished b-cell mass are hallmarks of the disease. We have previously shown that TLR2 tolerization by chronic treatment of a TLR2 agonist, Pam3CSK4, inhibits the development of diabetes in non-obese diabetes (NOD) mice. However, for the treatment of clinically overt T1D, critically reduced b-cell mass should be replenished in addition to immune tolerization. Dipeptidyl peptidase 4 (DPP4) inhibitor is a new class of anti-hyperglycemic agent that has beneficial effects on b-cell mass. We investigated if a combined treatment with DA1229, a new DPP4 inhibitor, and Pam3CSK4 could reverse established T1D. In early-onset diabetic NOD mice model, we observed long-term recovery from diabetes by chronic administration of Pam3CSK4 in combination with DA1229, which was accompanied by a significant increase in b-cell mass. The number of replicating b-cells and that of small b-cell unit representing b-cell neogenesis were significantly increased in mice treated with Pam3CSK4+DA1229. Diabetogenic T cell priming by dendritic cells and upregulation of costimulatory molecules after ex vivo stimulation were reduced in mice treated with Pam3CSK4 + DA-1229, indicating dendritic cell tolerance. These data demonstrate a combined treatment with Pam3CSK4+DA1229 inhibits the further progression of T1D and is able to reverse early-onset T1D, suggesting the potential therapeutic value of a combination therapy targeting the TLR2 blockade and DPP4 inhibition.