Background <p>Gastrointestinal stromal tumors (GIST) frequently develop secondary resistance to imatinib, which represents a major obstacle to achieving durable clinical benefit. However, the molecular mechanisms underlying acquired resistance remain poorly understood. Nestin, a cytoskeletal protein, has been implicated in tumor progression and cellular stress responses, but its role in imatinib-resistant GIST has not been fully elucidated.</p> Methods <p>Patient tumor specimens, imatinib-resistant GIST cell lines, and xenograft mouse models were analyzed to evaluate Nestin expression. Functional studies were performed using RNA interference–mediated silencing of Nestin. Mitophagy flux assays were conducted to evaluate mitochondrial quality control. The effects of Nestin modulation on PINK1 stability, mitophagy activity, mitochondrial integrity, and imatinib sensitivity were evaluated both in vitro and in vivo. Clinical correlations between Nestin expression, therapeutic response, and prognosis were also analyzed.</p> Results <p>Nestin expression was significantly upregulated in imatinib-resistant GIST across patient samples, resistant cell lines, and xenograft models. Mechanistically, Nestin stabilized PINK1, thereby enhancing PINK1-dependent mitophagy and preserving mitochondrial integrity under imatinib-induced stress. RNA interference suppression of Nestin reduced PINK1 accumulation, impaired mitophagy, increased mitochondrial damage, and restored imatinib sensitivity in vitro and in vivo. Clinically, elevated Nestin expression was associated with poor therapeutic response and unfavorable prognosis.</p> Conclusions <p>The Nestin–PINK1 axis is a critical driver of imatinib resistance in GIST. Targeting Nestin-mediated mitophagy may represent a promising therapeutic strategy to overcome imatinib resistance and improve clinical outcomes in patients with GIST.</p> Graphical Abstract <p></p>