Objective <p>To investigate the protective effect of cold-pressed flaxseed oil on oxidative damage in HepG2 cells and its mechanism.</p> Methods <p>HepG2 cells were cultured and divided into blank group, model group and cold-pressed flaxseed oil groups (low, medium and high dose). The effects of cold-pressed flaxseed oil on cell survival rate, levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and activities of antioxidant enzymes in the H₂O₂-induced oxidative stress injury model were determined.</p> Results <p>The results showed that cold-pressed flaxseed oil significantly alleviated the cell growth inhibition induced by H₂O₂, significantly decreased the levels of intracellular ROS and MDA, and increased the activities of superoxide dismutase, catalase and glutathione peroxidase (<i>P</i> &lt; 0.05). Transcriptomic analysis identified 32 differentially expressed genes (DEGs). GO analysis found that these DEGs were mainly involved in cellular processes, biological regulation, metabolic processes and transporter activities. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were mainly involved in mammalian target of rapamycin, mTOR signaling pathway and MAPK signaling pathway.</p> Conclusion <p>Cold-pressed flaxseed oil effectively protected HepG2 cells from oxidative damage, possibly by regulating the expression of the genes related to mTOR signaling pathway and MAPK signaling pathway.</p>