BACKGROUND: High-mobility group box 1 (HMGB1) plays various roles depending on its subcellular localization. Extracellular HMGB1 interacts with receptors, such as toll-like receptor 4 and receptor for advanced glycation end products (RAGE), promoting cell proliferation, survival, and migration in cancer cells. It also increases the expression of programmed death-ligand 1 (PD-L1) in cancer cells by binding to RAGE. However, the effect of intracellular HMGB1 on the regulation of immune checkpoints such as PD-L1 has not been well characterized. In this study, we aimed to investigate the effects of intracellular HMGB1 on PD-L1 expression in breast cancer cells. METHODS: Human and mouse triple-negative breast cancer cells, MDA-MB-231 and 4T1, along with HMGB1-deficient mouse embryonic fibroblast cells, were cultured. HMGB1 overexpression was achieved using a Myc-tagged plasmid, while siHMGB1 constructs were used for gene silencing. Quantitative reverse-transcriptase PCR and western blot analysis were performed to assess gene and protein expressions. Confocal imaging, immunoprecipitation, and proximity ligation assays were used to investigate HMGB1 localization and Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) interactions. In vivo experiments were performed using tumor-bearing mice treated with STAT3 and HMGB1 inhibitors. Statistical analyses were performed using Student's t-tests, one-way analysis of variance, Pearson's correlation, and Kaplan-Meier survival analysis, with significance set at p < 0.05. RESULTS: In breast cancer cells, HMGB1 translocation from the nucleus to the cytoplasm increased the JAK2-STAT3 interaction and induced STAT3 phosphorylation, leading to increased STAT3 target signaling, including the epithelial-mesenchymal transition (EMT) phenotype and PD-L1 expression. Inhibition of nucleo-cytoplasmic translocation of HMGB1 decreased STAT3 phosphorylation and PD-L1 expression. Furthermore, HMGB1 enhanced breast cancer cell migration, invasion, and EMT, contributing to tumor growth in an in vivo mouse model that were mitigated by the HMGB1-targeted approach. CONCLUSIONS: These findings underscore the critical role of intracellular HMGB1 in modulating PD-L1 expression via the JAK2-STAT3 signaling pathways in breast cancer and suggest that targeting HMGB1 translocation is a promising strategy for breast cancer treatment.