Following kidney transplantation, donor-specific hyporesponsiveness (DSH) may develop, defined as a lowered response of alloreactive T cells, specifically directed to donor Ag. This study aimed to characterize the nature of DSH through multiparameter flow cytometric assays measuring changes in phenotype and function of donor-reactive T cells after transplantation. This study characterized donor-reactive T cells, identified by CD137 expression, from the peripheral blood of stable human kidney transplant recipients (n = 47) before, at 3–5 y after, and >5 y after transplantation. The phenotype (T cell subset, differentiation status, and transcription factor expression) and function (proinflammatory cytokine production) of CD4+ and CD8+ donor-reactive CD137+ T cells was evaluated by both supervised and unsupervised analyses. Results demonstrated a decline in CD4+ donor-reactive T cells within the first 3–5 y after transplantation. Predominantly, the population of effector memory T cells capable of producing two or more proinflammatory cytokines was affected. This decline was strongly correlated with reduced proliferation of CD4+ T cells to donor Ag. The donor-reactive CD8+ T cells declined substantially only after >10 y. The frequency of T cells reactive to unrelated alloantigens did not alter significantly after transplantation, excluding an aspecific effect of immunosuppressive medication. After transplantation, an increase in donor Ag-induced apoptosis was found, specifically within the donor-reactive CD4+ memory T cell subsets. In conclusion, a significant decrease in donor-reactive polyfunctional effector memory CD4+ T cells underlies the development of DSH in kidney transplant recipients, which is likely mediated by specific activation-induced cell death.