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Breast cancer (BRCA) is one of the most common cancers in women. Copper (Cu) is an essential trace element implicated in many physiological processes and human diseases, including BRCA. In this study, we performed bioinformatics analysis and experiments to determine differentially expressed copper homeostasis-associated genes in BRCA. Based on two Gene Expression Omnibus (GEO) datasets, the copper homeostasis-associated gene, prion protein (PRNP), a highly conserved ubiquitous glycoprotein, was significantly down-regulated in BRCA compared to normal tissues. Moreover, PRNP expression predicted a better prognosis in BRCA patients. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that PRNP was potentially linked with several cancer-associated signaling pathways, including regulation of inflammatory response and oxidative phosphorylation. To validate the biological functions of PRNP, we overexpressed PRNP in BRCA cell lines, MDA-MB-231 and BT-549. CCK8 assay showed that PRNP overexpression significantly increased the sensitivity of gefitinib in BRCA cells. Overexpression of PRNP resulted in increased reactive oxygen species (ROS) production upon gefitinib treatment and ferroptosis selective inhibitor, ferrostatin-1 attenuated the enhanced ROS production effect of PRNP in BRCA cells. PRNP expression was positively correlated with macrophages, Th1 cells, neutrophils, and B cells, while negatively correlated with NK CD56 bright cells and Th17 cells in BRCA. Single-cell analysis showed that PRNP was highly expressed in M1 phenotype macrophages, essential tumor-suppressing cells in the tumor stroma. Therefore, our findings suggest that PRNP may participate in ROS-mediated ferroptosis and is a potential novel therapeutic target of chemotherapy and immunotherapy in BRCA.