Introduction: Cardiac transplantation provides a cure for end-stage heart diseases. Achieving long-term cardiac allograft survival without continuously global immunosuppression is a highly desirable goal because long-term immunosuppression causes serious side effects. This study is to induce long-term cardiac allograft survival without long-term conventional immunosuppression via treatments with Tanshinol, a major compound derived from traditional Chinese medicine, Danshen, which has been shown to improve tissue microcirculation and reduce inflammation. Hypothesis: Tanshinol alone, or in combination with mTOR inhibitor rapamycin, prolongs cardiac allograft survival. Methods: In this study, C57BL/6 mice were transplanted with a Balb/C heart and treated with Tanshinol (5mg/kg/day i.p. on days 0, 2, 4, 6, 8, 10 and 14) and/or rapamycin (1 mg/kg/day for 3 weeks). In vivo CD4+FoxP3+ Treg numbers from draining LNs and graft-infiltrating cells were quantified by FACS 3 weeks after transplantation. Results: We found that Tanshinol alone did not extend cardiac allograft survival compared to control group (MST = 8 vs. 7 days, n=7-8) while rapamycin delayed the rejection significantly (MST = 28 vs. 7 days, n=6-7, P<0.05). Interestingly, treatments with both Tanshinol and rapamycin further prolonged allograft survival (MST = 63 versus 28 days, n=7-8, P<0.05). Moreover, Tanshinol did not increase CD4+FoxP3+ Treg numbers in draining LN (DLN) (2.5+/-0.4 vs 2.2+/-0.3, x10000, P>0.05) while rapamycin did so (3.9+/-0.5 vs 2.2+/-0.3, P<0.05). In contrast, Tanshinol increased Treg number in grafts (13.5+/-0.9 vs 7.4+/-0.6, P<0.05) while rapamycin did not (8.1+/-0.7 vs 7.4+/-0.6, P>0.05). Tanshinol plus rapamycin augmented Treg numbers in both DLNs and grafts compared to untreated group. Thus, our studies reveal that Tanshinol and rapamycin synergistically prolong cardiac allograft survival without long-term immunosuppression by promoting Treg generation and migration to LNs and allografts. Conclusion: Our data, for the first time, demonstrate that Tanshinol synergizes with rapamycin to prolong cardiac allograft survival via enhancing both generation and migration of Tregs.