A previous paper has reported that blockade of NKG2D was effective in protecting allograft in murine models of cardiac transplantation, but the mechanism of NKG2D blockade on attenuated cardiac allograft vasculopathy (CAV) was still unknown. In our current study, we found that wild-type recipients treated with anti-NKG2D monoclonal antibody (mAb) plus cytotoxic T lymphocyte antigen (CTLA)-4-immunoglobulin (I)g showed prolonged allograft survivals (>90 days, P < 0·001) significantly and attenuated CAV. These in-vivo results correlated with reduced alloantibody production, low expression of interleukin (IL)-17 and IL-6, while infiltration of regulatory T cells increased. IL-6 administration induced shorter allograft survival and higher CAV grade in CTLA-4–Ig plus anti-NKG2D mAb-treated recipients, whereas IL-17 had no significant effect on allograft survival and CAV grade in CTLA-4–Ig plus anti-NKG2D mAb-treated recipients. Furthermore, the prolonged allograft survival induced by NKG2D blockade was abrogated partially with depletion of regulatory T cells. In conclusion, blockade of NKG2D combined with CTLA-4–Ig attenuated CAV and this effect was associated with lower alloantibody production, inhibited IL-6 expression and enhanced expansion of regulatory T cells.