Abstract: To investigate the crucial pathways and differentially expressed genes following the action of Ornithogalum saundersiae Saponins (OSW-1) on liver cancer cells (HEP3B), this study utilized transcriptome sequencing technology to examine the gene expression data of HEP3B cells treated with OSW-1. By precisely analyzing the transcripts from the measured sequence information, differentially expressed genes related to cell survival were screened to identify core genes. Furthermore, experiments were conducted to verify whether OSW-1 could treat hepatocellular carcinoma by regulating the core targets. Results showed that 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. In 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.