Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho‑mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.