The immune mechanism underlying Hashimoto's thyroiditis (HT) remains unclear. CD26, also known as dipeptidyl peptidase 4 (DPP-4), is a multifunctional molecule involved in the pathophysiology of autoimmune diseases. This study aimed to investigate the role of CD26 in the pathogenesis of HT. Peripheral blood was drawn from 20 healthy controls and 31 HT patients (19 mild HT patients and 12 severe HT patients). Plasma sCD26 concentrations were measured by ELISA, and sCD26 enzymatic activity was assessed using a luciferase-based assay. The expression levels of membrane-bound CD26 were analyzed by flow cytometry. Plasma sCD26 concentrations were lower in HT patients than in healthy controls, although the difference in sCD26 concentrations between the two groups did not reach statistical significance (P = 0.07). The percentages of CD8+ T cells and Tc1 cells with CD26 expression were decreased in HT patients compared with those in healthy controls, and the mean fluorescence intensity (MFI) values of CD26 on CD8+ T cells and Tc17 cells in HT patients were significantly lower than in healthy controls (P < 0.05). In HT patients, the expression of CD26 on CD8+ T cells and Tc subsets was decreased in the hypothyroidism group compared with that in the euthyroid group (P < 0.05). These results suggest that the sCD26 concentrations and membrane-bound CD26 levels on CD8+ T cells are aberrant in HT and that the reduced CD26 expression may be involved in the progression of HT.