Objective To investigate the effect of stimulants on the expression of T cell activation markers (CD69, CD38, HLA-DR) and a nuclear proliferation marker ( Ki67), and provide the basis for the study of T cell function after activation. Methods Peripheral blood was collected from healthy rhesus monkeys, then peripheral blood mononuclear cells ( PBMCs ) were separated and regular doses of PMA + Ionomycin, α-CD2 / α-CD3 / α-CD28, and phytohemagglutinin-P (PHA-P)+interleukin-2 ( IL-2) were added. Expression of CD4+ T cells and CD8⁺T cell surface markers, CD69, CD38, HLA-DR, and Ki67, were detected at different time points. Results Flow cytometry showed that when the stimulant, α-CD2 / α-CD3 / α-CD28, acted for 72 hours, expression of CD69 and Ki67 on CD4⁺T cells and CD8⁺T cells was higher compared with the other two stimulants, while the percentage of HLA-DR+ CD4⁺T cells and HLA-DR+ CD8⁺T cells and CD38⁺CD8⁺T cells was not different. Meanwhile, the percentage of CD38⁺CD4⁺T cells was not different between α-CD2 / α-CD3 / α-CD28 and PHA-P + IL-2 groups ( P> 0. 05). Expression of all four molecules was very low under PMA+Ionomycin stimulation. Conclusions CD2 / α-CD3 / α-CD28 had the best effect in activating T cells, followed by PHA-P+IL-2, and then PMA+Ionomycin. This provides the experimental basis for selecting appropriate stimulants on the basis of different experimental purposes.