Graft rejection is a critical cause of graft poor outcome even graft loss after transplantation, which is mainly associated with immune attack. Autophagy plays important roles in the cardiac ischemia/reperfusion process and its implicated in the pathogenesis of cardiac injury, although its role in the process is complicated. JQ1, a potent BET protein inhibitor, was reported to be capable of quenching hyper-inflammatory responses. Whether JQ1 has beneficial effect on transplant tolerance, and if so whether autophagy or immune suppression is involved in the protective effect has not been investigated. Our present study originally revealed that JQ1 treatment evidently prolonged allograft survival and well-preserved histological architecture in a mice model of cardiac transplantation. Intriguingly, we found that JQ1 efficiently induced an elevated protein expression of ATG5, ATG7, and LC3-II in cardiac allografts. Autophagy activation was suppressed by Bafilomycin A1 treatment based on the immunohistochemistry staining and immunoblot analyses of ATG5 and LC3-II. Furthermore, combined use of JQ1 with Bafilomycin A1 partially reversed the tolerogenic effect of JQ1 suggesting the involvement of autophagy signaling pathway in the process. Interestingly, JQ1 also decreased frequency of splenic Th1, Th2, Th17, and regulatory T cells as well and downregulated the expression of inflammatory cytokines such as IL-2, IL-6, IL-1beta, TNF-alpha, IFN-gamma, and IL-17. Taken together, these results indicate that JQ1 could significantly prolong allocardiac graft survival by potentiating myocardiac autophagy and inhibiting subsequent release of inflammatory cytokines. This unveiled mechanism may therefore provide novel insights into transplant tolerance induction.