Various memory cell populations existing before organ transplantation are believed to be important barriers to extending the graft survival time. Here, we report that arsenic trioxide (As 2 O 3 ), a common component of some Chinese traditional preparations, could obviously reduce the proliferation of splenic T cells in alloantigen-primed mice in vitro. Furthermore, we evaluated As 2 O 3 treatment alone or in combination with blocking monoclonal antibodies (mAb) against co-stimulatory molecules (LFA-1 and CD154) in the prevention of heart transplant rejection in alloantigen-primed mice. Alloantigen-primed mice were randomly divided into 4 groups of 6 animals each. The control group was given normal saline; the As 2 O 3 group received As 2 O 3 (5mg/kg/d) (i.p.) on days 0–10 post-transplantation; the Ab group received 100μg anti-LFA-1 mAb+250μg anti-CD154 mAb (i.p.) on the day of transplantation and 3 more times every alternate day and the As 2 O 3 +Ab group received a combined As 2 O 3 and Ab treatment protocol. The survival of the allografts was almost doubled among the As 2 O 3 group and Ab group compared with the control group (5, 5.6 vs 3.3days). A marked prolongation (16days) of graft survival was achieved by the combination protocol compared with the control group (3.3days; P<0.05). None of the treatment group showed side effects. Five days after transplantation, cardiac allografts, splenic T cells and serum were harvested for analysis. Compared with the control group, most of the treatment groups showed: (i) alleviation of allograft rejection in different levels; (ii) IFN-γ expression was reduced and TGF-β expression increased in both peripheral blood and within the grafts; (iii) the proportions of CD4 + or CD8 + memory T cells in the spleen of the recipients were significantly reduced while the expression of regulatory T cells (Tregs) was enhanced; (iv) the alloresponses of memory T cells were inhibited, and levels of alloantibodies in recipients' sera were also decreased. Among all these changes, the As 2 O 3 +Ab group showed the most significant changes. Our study highlights an obvious synergistic action of As 2 O 3 when combined with co-stimulatory molecules blockade in prolonging the survival of cardiac allografts in alloantigen-primed mice.