Abstract: Objective: To explore the crucial pathways and genes that are expressed differently when hepatocellular carcinoma cells (HEP3B) were treated with Ornithogalum saundersiae Saponins (OSW-1), using transcriptome sequencing. Methods: The gene expression data in HEP3B treated with OSW-1 were obtained through transcriptome sequencing technology. The transcripts were analyzed precisely based on the sequence information measured, and genes with differential expression related to cell survival were identified to discover the core genes. Through experimental methods, it was intended to confirm whether OSW-1 could treat hepatocellular carcinoma by targeting core genes. Results: The MTT assay revealed a significant reduction in HEP3B cell activity following OSW-1 treatment, which was dependent on the concentration. A total of 1381 genes showed significant differences in expression as a result of transcriptome sequencing, with a majority being involved in the TNF signaling pathway. Additionally, immunofluorescence and Western blot analysis indicated that the levels of p-PI3K, p-AKT, and p-NF-κB proteins were reduced in HEP3B cells after treatment with OSW-1. Conclusion: OSW-1 may suppress inflammation and reduce HEP3B cell activity by modulating inflammatory signaling pathways, suggesting its potential as a drug for liver cancer treatment and providing an experimental foundation for the development of healthy foods aimed at liver protection.