SummaryBoth chemokine receptor 5 (CCR5) blockade and rapamycin (rapa) are effective in modulating transplant immunity and led to prolonged allograft survival, yet a great many grafts were ultimately lost to acute rejection. In this study we examined the inhibition of CCR5 in combination with the treatment with rapa in cardiac transplantation. Fully major histocompatibility complexmismatched murine cardiac allograft models were randomized to five groups. They were administered with anti-CCR5 antibody or control antibody and rapa or phosphate-buffered saline (PBS), respectively. An additional group was treated with anti-CCR5 antibody, rapa and anti-CD25 antibody. Allograft rejection was investigated by flow cytometric analyses and enzyme-linked immunospot assay. Allografts treated with anti-CCR5 antibody plus rapa showed significantly prolonged survival (83 Ϯ 3 days, P < 0·001) compared with control antibody plus PBS-treated allografts (6 Ϯ 1 days). Treatment with anti-CCR5 monoclonal antibody (mAb) plus rapa inhibited significantly the progression of chronic rejction. Further analysis of donor hearts in the anti-CCR5 antibody plus rapa-treated group demonstrated increased infiltration of CD4 + CD25 + forkhead box P3 + regulatory T cells, and depletion of CD25 + cells resulted in acute rejection of allografts in 18 Ϯ 1 day. CCR5 blockade in combination with rapa is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated by CD25 + T cell recruitment and control of T lymphocyte proliferation.