High mobility group box 1 (HMGB1) is a non‑histone protein widely expressed in the nucleus of mammalian cells, and it can be released by both immune and tumor cells. In the extracellular context HMGB1 can act as a proinflammatory mediator and boosting cancer progression. High HMGB1 mRNA expression levels are usually observed in various malignant diseases, including breast cancer (BC). Several studies have demonstrated the potential clinical value of HMGB1 in BC diagnosis and therapy. The present data, using in vitro protocols and molecular technologies, demonstrated the presence of HMGB1 in organoids derived from patients with BC with significantly elevated expression correlating with poorer prognosis. By blocking the activity of HMGB1 protein, cell cycle arrest and induction of apoptosis was observed in ex vivo 3D organoids, suggesting a potential antitumor effect. The localization by immunofluorescence, of HMGB1, β‑catenin and NF‑kB in organoids and the subsequent inhibition of the entire molecular pathway by switching off HMGB1 signaling suggests that there is a crosstalk between these molecules demonstrating their involvement in inflammation and inflammation‑associated diseases such as cancer. The current results aim to investigate the role of HMGB1 in BC progression and find innovative applications based on HMGB1 as a therapeutic target and early disease biomarker.