Infusion of ethylene carbodiimide-fixed donor splenocytes (ECDI-SPs) is an effective method to induce donor-specific protection to allografts. However, the ischemia reperfusion (I/R) injury during transplant leads to abundant of pro-inflammatory cytokines, which negates the effect of ECDI-SPs. Therefore, suppressing pro-inflammatory cytokine secretion while promoting anti-inflammatory cytokine release would enhance the graft protective efficacy of ECDI-SPs. In this study, we aimed to determine the effect of ECDI-SPs combined with a short course of cordycepin (an anti-inflammatory agent) on the long-term outcomes of mice cardiac allografts. Our results demonstrated that ECDI-SPs combined with cordycepin significantly promoted mice cardiac allograft survival compared with ECDI-SPs monotherapy. This effect was accompanied by decreased production of pro-inflammatory cytokines (IL-1β, IL-6, IL-17 and TNFα), increased secretion of anti-inflammatory cytokines (IL-10 and TGFβ), inhibition of Th17 and expansion of Tregs, and prevention of I/R injury. We concluded that cordycepin appeared to enhance the effect of modulating cytokine profile and regulate the Teff:Treg balance so as to strengthen the graft protective effect of ECDI-SPs. Our study of ECDI-SPs combined with cordycepin may provide a promising approach for prolong allograft survival.