<p>Adriamycin (ADR) is a chemotherapy drug for breast cancer, and its resistance is a major obstacle in the clinical treatment of breast cancer. O-GlcNAcylation is a post-translational modification that impacts chemotherapy resistance in cancers. This present study aims to investigate the mechanism of O-GlcNAcylation-mediated ADR resistance in breast cancer. Cell viability, proliferation, and apoptosis were performed to evaluate ADR resistance in breast cancer cells. O-GlcNAcylation, OGA and OGT levels in patients, and breast cancer cells resistant to ADR or not were detected by western blot and quantitative real-time PCR. Glycolysis of ADR-resistant cells was evaluated by measurement of glucose and lactic acid levels, and extracellular acidification rate and oxygen consumption rate. The underlying mechanism was explored by western blot and pathway enrichment analysis. Effects of OGT in vivo were assessed by xenograft tumor model. Results showed that OGT protein and mRNA levels were increased in MCF-7R and BT-549R cells and tumors of ADR-resistant patients with breast cancer. Moreover, O-GlcNAcylation was increased in ADR-resistant breast cancer cells. OGT knockdown inhibited glycolysis and O-GlcNAcylation and protein level of MDM4 at S96 site. Notably, MDM4 overexpression restored glycolysis in MCF-7R and BT-549R cells inhibited by OGT knockdown. Additionally, OGT knockdown inhibited tumor growth in vivo. Collectively, this study demonstrated that OGT promote breast cancer resistant to ADR through facilitating glycolysis in breast cancer cells by O-GlcNAcylation on MDM4. This study may provide a target for overcoming ADR resistance in breast cancer.</p> Graphical Abstract <p>OGT is upregulated in ADR-resistant breast cancer cells and enhances MDM4 protein stability through O-GlcNAcylation of MDM4, thereby promoting cell proliferation and glycolysis while inhibiting apoptosis in ADR-resistant breast cancer cells.</p> <p></p>