BACKGROUND Although 5-Flourouracil(5-FU) is used as the first-choice treatment for advanced hepatocellular carcinoma (HCC), it is associated with acquired and intrinsic resistance. Hyperactivation of mTOR signaling has been linked to tumorigenesis and chemoresistance in HCC. The aim of this study was to evaluate and compare the antitumor effects of mTORC1 inhibitor everolimus and mTORC1/2 inhibitor AZD8055 and to examine the interaction between 5-FU and mTORC1/2 inhibitor in HCC. MATERIAL AND METHODS Using cultured HCC cells and mouse xenograft, the antitumor effects of everolimus and AZD8055 were analyzed as mono- and combination therapy with 5-Flourouracil. RESULTS TSC2-deficient HCC cell lines were more sensitive to everolimus and AZD8055. AZD8055, but not everolimus, potently prevented cells from transitioning from G1 phase to S phase in TSC2-high-expressing HCC cells. AZD8055 reduced phosphorylation of both mTORC1 and mTORC2 substrates. In contrast, everolimus reduced the phosphorylation of mTORC1 substrates, but increased the phosphorylation of AKT. Notably, AZD8055, but not everolimus, synergistically enhanced the efficacy of 5-FU via reversing 5-FU-induced upregulation of P-glycoprotein (P-gp). The synergistic antitumor effect of AZD8055 and 5-FU was also observed in a HCC xenograft mouse model. CONCLUSIONS TSC2 in HCC is a promising efficacy-predicting biomarker for the treatment of mTORC1/2 inhibitor. AZD8055 showed stronger antitumor activity than everolimus in TSC2-high-expressing HCC cells. Moreover, the combination of mTORC1/2 inhibitor with 5-FU appears to be a promising option for HCC patients refractory to chemotherapy.