B7-H3, an immune checkpoint molecule, is overexpressed in various solid cancers, correlating with negative prognosis and poor clinical outcomes. However, its regulatory mechanisms in aggressive cancers and cancer stem cells (CSCs), which contribute to cancer formation, progression, chemoresistance, and recurrence, remain unclear. To elucidate the mechanisms regulating B7-H3 expression in breast CSCs, we employed DNA affinity purification-mass spectrometry (DAP-MS) with presumptive promoter regions of B7-H3 and identified several proteins binding to that promoter regions in CSC specific manner. Subsequent validation studies identified several potential transcription factors, including DNA damage-binding protein 1 (DDB1), XRCC5, PARP1, RPA1, and RPA3, for their significant impact on B7-H3 expression. In vitro inhibition of DDB1 with its known inhibitor, nitazoxanide, resulted in a marked decrease in B7-H3 expression, suppressing tumor sphere formation and cell migration in breast CSCs. Our findings reveal a transcription factor-mediated regulatory mechanism for B7-H3 in CSCs through proteomic analysis and highlight the critical role of these transcription factors in regulating B7-H3 expression. These results may elucidate underlying mechanisms, paving the way for the development of new immunotherapeutic strategies against breast cancer.