Abstract: Objective: To investigate the therapeutic effects and potential mechanisms of telocinobufagin in hepatocellular carcinoma(HCC). Methods: Human HCC cell lines SK-HEP-1 and Huh-7 were used as the research subjects. The effect of telocinobufagin on cell viability was assessed by cell counting kit-8(CCK-8) assay, and that on cell proliferation capacity by colony formation assay. The apoptosis was evaluated using Hoechst 33258 staining and flow cytometry. Mitochondrial membrane potential changes were measured with JC-1 fluorescent probe staining. Cell cycle distribution was analyzed by flow cytometry. Transcriptome sequencing(RNA-seq) and enrichment analysis were performed to clarify the effect of telocinobufagin on the gene expression profile of cells, and reverse transcription quantitative real-time PCR(RT-qPCR) and western blotting were used to verify the expression changes of related genes and proteins after drug treatment. An in vivo xenograft tumor model was established in KM mice to assess the safety and anti-tumor efficacy of the drug. Results: Telocinobufagin significantly inhibited the viability and proliferation of HCC cells, reduced mitochondrial membrane potential, induced apoptosis, and caused cell cycle arrest(P<0.01). RNA-seq indicated marked downregulation of the protein expression in the mTOR signaling pathway. Intracellular cholesterol content was decreased, accompanied by reduced expression of downstream cholesterol synthesis-related genes(P<0.01). Results of the tumorigenesis assay in mice demonstrated that telocinobufagin notably suppressed tumor growth in vivo(P<0.05). Conclusion: Telocinobufagin may exert anti-HCC effects by inducing apoptosis, arresting the cell cycle in HCC cells, and inhibiting cholesterol synthesis through modulation of the mTOR signaling pathway.