Background <p>A complex interplay between oxidative stress, mitochondrial dysfunction, and apoptosis contributes to the pathogenesis of neurodegenerative diseases (ND). Annexin A5 (ANXA5) is a calcium-binding multifunctional protein. This study aimed to evaluate the possible protective effects of ANXA5 protein against H₂O₂-induced cell death, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) dissipation, and expression of <i>Bax</i>, <i>Bcl2,</i> and <i>NRF2</i> in SH-SY5Y cells.</p> Methods <p>SH-SY5Y cells were treated with different concentrations of H₂O₂, and cell viability was determined using an MTT assay. MMP dissipation and ROS accumulation were measured using rhodamine 123 and DCF-DA flow cytometry. The DNA fragmentation assay and real-time PCR were conducted to determine the effects of the treatments on genomic DNA damage and gene expression.</p> Results <p>H₂O₂ reduced the viability of SH-SY5Y cells in a dose-dependent manner (IC₅₀ = 550µM). Moreover, H₂O₂ at the IC₅₀ concentration induced ROS accumulation, MMP loss, increased <i>Bax</i> expression, and reduced <i>NRF2</i> expression in the SH-SY5Y cells. ANXA5 reversed the cytotoxic effects of H₂O₂ on the SH-SY5Y cell viability, ROS accumulation, MMP loss, and gene expression.</p> Conclusion <p>ANXA5 can protect the SH-SY5Y cells against H₂O₂-induced oxidative stress, apoptosis, and mitochondrial dysfunction.</p>