Objectives <p>The purpose of this study is to examine the molecular pathways underlying breast cancer and the function of Notch-1 in tumor-associated macrophages (TAMs).</p> Methods <p>To identify Notch-1 as the most differentially expressed critical molecule in tumor-associated macrophages (TAMs) and to predict its downstream regulatory network, bioinformatics research was first carried out utilizing the GSE33447 and TCGA datasets. A TAM–tumor cell interaction model was then created in vitro by co-culturing THP-1 monocytes with MCF-7 breast cancer cells after inducing them into macrophages using PMA (Phorbol 12-myristate 13-acetate). Notch-1 overexpression (OE) and knockdown (KD) THP-1 cell lines were created. The expression of inflammatory cytokines, TAM phagocytic activity, and their impact on MCF-7 cell proliferation, apoptosis, and migration/invasion were evaluated by flow cytometry, ELISA, Western blot, qRT-PCR, scratch assay, and Transwell invasion assay; immunofluorescence was used to confirm macrophage differentiation. To create a xenograft tumor model, THP-1-derived macrophages from various treatment groups were co-implanted subcutaneously with MCF-7 cells into BALB/c nude mice. To confirm that Notch-1-regulated TAM function inhibits tumor development, histological examination was carried out.</p> Results <p>The EZH2/BRD4/PLK1/FOXM1/USP22/PU.1 signaling axis is closely associated with Notch-1, a crucial regulator in TAMs, according to bioinformatics study. Notch-1 overexpression in TAMs dramatically increased the expression of inflammatory cytokines (IL-1β, IFN-γ) and phagocytic activity, which inhibited the migration and proliferation of MCF-7 breast cancer cells, according to in vitro experiments, whereas Notch-1 knockdown had the opposite effect. Notch-1 increased EZH2, BRD4, PLK1, FOXM1, USP22, and PU.1, creating a positive feedback regulatory network that encourages the pro-inflammatory polarization of TAMs, according to further mechanistic research. Co-implantation of Notch-1-overexpressing TAMs with MCF-7 cells dramatically reduced tumor development and improved macrophage infiltration and phagocytic activity inside tumor tissues, according to in vivo investigations. In summary, Notch-1 in TAMs suppresses tumor growth via controlling the EZH2/BRD4/PLK1/FOXM1/USP22/PU.1 axis to trigger phagocytosis and inflammatory reactions.</p> Conclusions <p>Notch-1 in TAMs inhibits the growth of breast cancer by controlling the EZH2/BRD4/PLK1/FOXM1/USP22/PU.1 signaling pathway, which causes inflammation and phagocytosis.</p>