This study aims to screen molecular targets of bufalin in promoting apoptosis of human hepatocellular carcinoma cells.Methods:The effect of bufalin on apoptosis and gene expression of human hepatocellular carcinoma PLC/PRF/5 cells was investigated.To be specific，the PLC/PRF/5 cells were treated with different concentration of bufalin for 24 h，48 h，and 72 h，respectively，and the proliferation and apoptosis of the cells were detected.Illumina high-throughput sequencing of RNA was performed to observe the changes of gene expression in the cells treated with bufalin，and the bioinformation of related genes was analyzed.Results:Bufalin inhibited the proliferation and induced the apoptosis of PLC/PRF/5 cells.It induced the expression of different genes.Gene Ontology(GO) term analysis showed that bufalin influenced cell cycle，apoptosis，and other pathways.Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis suggested that calpain was significantly up-regulated in calcium-induced cell death pathway.Conclusion:Bufalin remarkably promotes the apoptosis of human hepatocellular carcinoma cells through multiple targets in related pathways.