Background <p>The importance of fatty acids in human health and their potential in treating various brain diseases is increasingly acknowledged. Research indicates that ultra-long-chain fatty acids adversely affect dietary habits, while omega (<i>ω</i>)-3 polyunsaturated fatty acids confer health benefits. Eicosapentaenoic acid (EPA), an <i>ω</i>-3 polyunsaturated fatty acid, manifests diverse protective activities, including anti-oxidative effects and the attenuation of brain diseases. Previous studies have suggested that EPA can alleviate oxidative stress and forestall diseases stemming from oxidative damage. Nevertheless, EPA’s precise antioxidant mechanism and signaling pathway in human astrocytes remain elusive. To address this knowledge gap, we established an H₂O₂-induced oxidative damage model in Gibco^® Human Astrocytes (GHA cells) and elucidated the underlying mechanisms and signaling pathways.</p> Methods and results <p>Our assessments included cell viability through the CCK-8 assay, morphological examination via microscopy, ROS quantification using the DCFH-DA fluorescent probe, GSH content evaluation with the CMF-DA fluorescent probe, and protein expression analysis for antioxidant and apoptotic markers through Western blotting. The results showed that pretreatment with 3 µM of EPA countered the cytotoxicity, ROS production, and GSH depletion caused by H₂O₂ (250 µM) in GHA cells. Additionally, EPA pretreatment effectively reduced the cytotoxicity and oxidative stress resulting from H₂O₂ by modulating the Nrf2/HO-1/NQO1 and Bax/Bcl-2/caspase-9/caspase-3 signaling pathways in GHA cells.</p> Conclusion <p>These findings enhance our understanding of EPA’s antioxidant mechanisms in the oxidative stress model of human astrocytes, illuminate the interplay between antioxidant and apoptotic signals, and offer promise for exploring potential preventive and therapeutic interventions for brain diseases.</p>