Background: Acute graft rejection mediated by alloreactive memory CD4(+) T cells is a major obstacle to transplantation tolerance. It has been reported that CD8(+) T regulatory cells (Tregs) have the ability to induce graft tolerance by restraining the function of activated CD4(+) T cells, but not including memory T cells. The aim of this study is to elucidate the effect of CD8+ Tregs on alloreactive memory CD4(+) T cells. Methods: We detected Qa-1 expression and performed proliferative assay on memory CD4(+) T cells. All memory CD4(+) T cells were purified from mice receiving skin allografts. We performed inhibitory and cytotoxic assays on CD8(+) Tregs, which were isolated from a T cell vaccination mouse model, and IL-2, IL-4, IL-10 and IFN-gamma levels were measured in co-culture supernatants by ELISA. To confirm CD8(+) Tregs inhibition of memory CD4(+) T cells in-vivo, we utilized a murine model of cardiac allograft transplantation. Results: Memory CD4(+) T cells mediated acute allograft rejection, and CD8(+) Tregs suppressed the proliferation of memory CD4(+) T cells. In vitro, memory CD4(+) T cells were inhibited and lysed by CD8(+) Tregs. There was a positive correlation between IFN-gamma levels, and cell lysis rate induced by CD8(+) Tregs. In-vivo studies demonstrated CD8(+) Tregs prolonged graft survival times, by inhibiting CD4(+) memory T cells, through a Qa-1-peptide-TCR pathway. Conclusions: CD8(+) Tregs inhibit CD4(+) memory T cell-mediated acute murine cardiac allograft rejection, and further prolong graft survival times. These results provide new insights into immune regulation of organ rejection.