Objective · To reveal the mechanism of the effect of arsenic trioxide (ATO) on hepatocellular carcinoma (HCC) cells through metabolomics study. Methods · HCC cell line HepG2 was treated with ATO for 12 h, 24 h and 36 h respectively. The global metabolite profiles were monitoredmetabolomics analysis using GC/MS and LC/MS/MS. Results · A total of 307 certified metabolites were detected. Bioinformatics analysis showed that glycosylation and one-carbon metabolism were the main cellular functions that have been affected. Other affected cellular functions/pathways included mitochondrial function fatty acid β-oxidation, TCA cycle, pentose phosphate pathway (PPP) and glutathione metabolism. Conclusion · ATO treatment affects the main metabolomic pathway of HepG2, and inhibits cell growth and migration, then exacerbates oxidative stress to induce cancer cell apoptosis.