Overexpression of survivin, a member of the inhibitor of apoptosis protein family, has been confirmed as a poor prognosis factor in patients with hepatocellular carcinoma (HCC). Any treatment which aims to inhibit the expression of survivin may provide a new hope for HCC therapy. Liver transplantation (LT) has been demonstrated to provide the best treatment for patients with HCC since it removes both the tumor and the underlying cirrhotic liver, but the best treatment for HCC recurrence after LT needs further investigation. Sorafenib, a small molecule inhibitor of several tyrosine kinases and Raf kinases, has been used as the standard agent for the treatment of advanced HCC. Furthermore, sorafenib in combination with various immunosuppressants has been used for patients with recurrent HCC after LT with marginal outcome. In this study, we evaluated the cytotoxic effects of a novel survivin inhibitor, YM155, which has been investigated in several phase II clinical studies for the treatment of variant malignancies, plus rapamycin, everolimus, or tacrolimus in survivin-rich human HCC cell line HepG2 in vitro. YM155 at 100, 250, and 500 nM inhibited the proliferation of HepG2 after 72 hrs treatment. After 48-hr treatment, YM155 also elicited concentration-dependent cytotoxicity, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, with IC50 of around 200 nM. In addition, after treatment of HepG2 cells with 250 nM YM155 for 48 hrs, apoptosis detected by the presence of DNA fragments in the cytoplasm was observed; the expression of survivin, securin, and livin measured by Western blotting was down-regulated, unchanged, and up-regulated, respectively. Increased LC3-II expression is a marker for autophagy activation, which subsequently may lead to type II death. After one 48 hr-treatment with 250 nM YM155, the expression of LC3-II was increased. Autophagy inhibitor 3-methyladenine at 1 mM ameliorated YM155-induced cytotoxicity. In contrast, YM155 plus mTOR (mammalian target of rapamycin) inhibitor, rapamycin, enhanced the LC3-II expression. Cell viability after treatment with 1μM rapamycin and 250 nM YM155 for 48 hrs was 78.71% and 61.65%, respectively. But cell viability was dropped to 26.33% after 48 hrs treatment with 1 μM rapamycin plus 250 nM YM155. Cell viability after treatment with another mTOR inhibitor, everolimus, at 1μM alone was 80.4%, and the viability dropped to 40.07% after treatment with 1 μM everolimus plus 250 nM YM155 for 48 hrs. Calcineurin inhibitor tacrolimus alone elicited no cytotoxic effect on HepG2 cells 48 hrs after treatment. And no additive or synergistic cytotoxicitiy on HepG2 cells was observed after treatment with YM155 combined with tacrolimus for 48 hrs. In conclusion, YM155 plus rapamycin exerted the most potent and synergistic cytotoxicity through both type I death (apoptosis) and type II death (autophagy). YM155 when used with rapamycin may be an effective therapy for recurrent HCC after liver transplantation.