Purpose <p>This study aims to investigate the mechanisms underlying ANXA1 and HMGB1-regulated autophagy in relation to growth and radioresistance in thyroid cancer cells. This exploration seeks to provide new theoretical insights and potential therapeutic targets for clinical management of ¹³¹I-resistant thyroid cancer.</p> Methods <p>The expression levels of ANXA1 in thyroid cancer tissues and cells were assessed through immunohistochemical staining and Western blot analysis. The correlation between ANXA1 expression levels and resistance to ¹³¹I was evaluated using cell viability assays (CCK-8), apoptosis analysis and colony formation assays. Additionally, the relationship between ANXA1 and autophagy was investigated by employing mRFP-GFP-LC3 virus transfection in conjunction with confocal microscopy to assess autophagic flux, as well as through Western blotting. Proteomics analysis, co-immunoprecipitation (Co-IP) results, along with various in vitro and in vivo experiments further elucidated the relationship between ANXA1 and HMGB1.</p> Results <p>This study demonstrated for the first time that ANXA1 promotes the development of ¹³¹I resistance through the activation of autophagic flux. Proteomic analysis, co-immunoprecipitation (Co-IP) results, as well as various in vitro and in vivo experiments corroborated that ANXA1 activates autophagy by upregulating HMGB1 expression, thereby facilitating ¹³¹I drug resistance in thyroid cancer cells.</p> Conclusion <p>This study unveils a novel mechanism by which ANXA1 fosters drug resistance to ¹³¹I in thyroid cancer via regulation of the HMGB1-mediated autophagy pathway. This not only provides a new theoretical framework for understanding iodine resistance in thyroid cancer but also identifies a potential molecular target for developing combination therapy strategies aimed at treating iodine-refractory thyroid cancer.</p>