The aim of this study was to evaluate and determine the potential mechanisms of As₂O₃ in accelerated rejection mediated by alloreactive CD4⁺ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4⁺ memory T cells) was performed on Day 0, and As₂O₃ was administered to recipient mice from Day 0 to 10. As a result, As₂O₃ could reduce the proliferation of allo-primed CD4⁺ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As₂O₃ group was prolonged from 5.8 ± 0.7 to 14.2 ± 2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As₂O₃ treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4⁺ T cells, CD4⁺ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As2O3 as a novel immunosuppressant targeting CD4⁺ memory T cells.