CD26, acting as a co-stimulator 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 analysed in the different subsets of human peripheral blood T lymphocytes after antigen stimulation. Here, the percentage of CD4+ cells significantly increased and most of these cells were co-expressed with CD26, suggesting a close correlation of CD26 expression with the proliferation of CD4+ cells. Subsequently, after antigen stimulation, CD26 high-expressing cells (CD26high) were separated from CD26 low-expressing cells (CD26low) by magnetic cell sorting. We found that the percentages of cells secreting Th1-typical cytokines (IL-2, IFN-γ), Th17-typical cytokines (IL-6, IL-17, IL-22) or expressing Th17-typical biomarkers (IL-23R, CD161, CD196) in the CD26high group were markedly higher than in the CD26low group. In addition, a co-expression of CD26 with IL-2, IFN-γ, 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.