<p>Cancer stem cells (CSC) and the immunosuppressive microenvironments are key drivers of breast cancer (BC) progression and drug resistance. However, the molecular mechanisms by which One Cut Homeobox 2 (ONECUT2) governs CSC and the tumor immune microenvironment (TIME) remain largely unknown. Given the critical knowledge gap, we sought to investigate ONECUT2’s regulatory impact on CSC properties and TIME profiles using BC cell lines, animal models, and clinical specimens. Here, we demonstrated that inhibition of ONECUT2, a core transcription factor, potently drives CSC characteristics and reprograms the TIME to favor macrophage polarization to the M2-type, a tumor-promoting state. Mechanistically, ONECUT2 inhibition transcriptionally activated POU6F2, which subsequently triggered beta-catenin, thereby enhancing CSC properties and chemoresistance in BC. With respect to modulating the immune microenvironment, suppression of ONECUT2 governs macrophage polarization toward the immunosuppressive M2 phenotype. CCL28 is identified as a transcriptional target of ONECUT2 required for M2-type macrophage polarization, and CCR10 as a key receptor involved in this immune modulation. These findings highlight the critical involvement of ONECUT2 in modulating BC stemness via targeting the POU6F2-beta-catenin axis and managing macrophage polarization to M2 phenotype through the CCL28-CCR10 pathway. Our study suggests that ONECUT2 modulates cancer stemness and the TIME, and that targeting its downstream POU6F2-beta-catenin axis and CCL28-CCR10 pathway may provide an effective approach for BC treatment.</p>