CD26, acting as a costimulator of T cell activation, plays an important role in the immune system. However, the role of CD26 in the differentiation of T cell subsets, especially of new paradigms of T cells, such as Th17 and Tregs, is not fully clarified. In the present study, the role of CD26 in T cell differentiation was investigated in vitro. CD26 expression was analyzed in the different subsets of human peripheral blood T lymphocytes after solid-phase immobilized specific anti-CD3 mAb stimulation. Here, the percentage of CD4(+) cells significantly increased and most of these cells were coexpressed with CD26, suggesting a close correlation of CD26 expression with the proliferation of CD4(+) cells. Subsequently, after immobilized anti-CD3 mAb stimulation, CD26 high-expressing cells (CD26(high)) were separated from CD26 low-expressing cells (CD26(low)) by magnetic cell sorting. We found that the percentages of cells secreting Th1 typical cytokines (IL-2, IFN-gamma) and Th17 typical cytokines (IL-6, IL-17, and IL-22) or expressing Th17 typical biomarkers (IL-23R, CD161, and CD196) in the CD26(high) group were markedly higher than in those in the CD26(low) group. In addition, a coexpression of CD26 with IL-2, IFN-gamma, IL-17, IL-22, and IL-23R in lymphocytes was demonstrated by fluorescence microscopy. These results provide direct evidence that the high expression of CD26 is accompanied by the differentiation of T lymphocytes into Th1 and Th17, indicating that CD26 plays a crucial role in regulating the immune response.