Objective To investigate the mechanism of ferroptosis mediated by the bromodomain protein subfamily 4（BRD4）/nuclear factor-kappa B（NF-κB） signaling pathway in the malignant biological behavior of breast cancer. Methods Doxorubicin-resistant MDA-MB-231 cells（MDA-MB-231/DOX） were divided into control group（Con group），DOX group，and si-BRD4+DOX group. In the si-BRD4+DOX group，MDA-MB-231/DOX cells were transfected with si-BRD4 for 48 hours and were then treated with 1 μmol/L DOX for 24 hours. Colony formation assay and 5-ethynyl-2'-deoxyuridine（EdU） analysis were used to evaluate the proliferation ability of cells，and FerroOrange and liperfloo were used to measure the concentration of ferrous ion and the level of lipid peroxidation. A total of 15 female BALB/c-nu mice were randomly divided into control group，DOX group，and DOX+Si-BRD group，with 5 mice in each group，and a subcutaneous inoculation model of MDA-MB-231/DOX cells was established. The methods of qRT-PCR，Western blotting，and immunohistochemistry were used to measure the expression of the BRD4/NF-κB signaling pathway. Results Compared with the si-NC group，the si-BRD4#1 group and the si-BRD4#2 group had significant reductions in the number of cell clones，positive EDU staining，and glutathione level in MDA-MB-231 and BT549 cells（P<0.001），as well as significant increases in the level of ferrous ion，the level of reactive oxygen species（ROS），and oxidized glutathione/glutathione ratio（P<0.01）. Compared with the parental cells（MDA-MB-231），there were increases in the mRNA and protein expression levels of BRD4 in MDA-MB-231/DOX cells. Compared with the Con group，the DOX group had significant increases in the expression levels of IKβ-α，NF-κB，and BRD4 and the level of ROS（P<0.05） and significant reductions in the number of cell clones and positive EDU staining（P<0.05）； compared with the DOX group，the si-BRD4+DOX group had significant reductions in the expression levels of IKβ-α and NF-κB，the number of cell clones，and positive EDU staining（P<0.05） and a significant increase in the level of ROS（P<0.05）. Compared with the control group，the DOX group had significant reductions in tumor weight and volume（P<0.05） and a significant increase in the number of TUNEL-stained cells in tumor tissue（P<0.05）. In addition，compared with the DOX group，the BRD4+DOX group had further reductions in tumor weight and volume（P<0.001） and a significant increase in the number of TUNEL-stained cells（P<0.001）. Compared with the DOX group，the BRD4+DOX group had significant reductions in Ki-67，BRD-4，and NF-κB（P<0.01） and a significant increase in 4-hydroxynonenal（P<0.01） in tumor tissue. Conclusion BRD4 is an important drug resistance factor，which can inhibit ferroptosis induced by chemotherapy in breast cancer by promoting the activation of the NF-κB signaling pathway.