A defining feature of basal-like breast cancer, a breast cancer subtype with poor clinical prognosis, is the high expression of “proliferation signature” genes. We identified B-Myb, a MYB family transcription factor that is often amplified and overexpressed in many tumor types, as being highly expressed in the proliferation signature. However, the roles of B-Myb in disease progression, and its mammary-specific transcriptional targets, are poorly understood. Here, we demonstrated that B-Myb expression is a significant predictor of survival and pathological complete response to neoadjuvant chemotherapy in breast cancer patients. We also identified a significant association between the G/G genotype of a nonsynonymous B-Myb germline variant (rs2070235, S427G) and an increased risk of basal-like breast cancer [OR 2.0, 95% CI (1.1-3.8)]. In immortalized, human mammary epithelial cell lines, but not basal-like tumor lines, cells ectopically expressing wild-type B-Myb or the S427G variant showed increased sensitivity to two DNA topoisomerase IIα inhibitors, but not to other chemotherapeutics. In addition, microarray analyses identified many G2/M genes as being induced in B-Myb overexpressing cells. These results confirm that B-Myb is involved in cell cycle control, and that dysregulation of B-Myb may contribute to increased sensitivity to a specific class of chemotherapeutic agents. These data provide insight into the influence of B-Myb in human breast cancer, which is of potential clinical importance for determining disease risk and for guiding treatment.