Background Galectin-3 (Gal3) plays diverse roles in cancer initiation, progression, and drug resistance depending on tumor type characteristics that are also associated with cancer stem cells (CSCs). Recurrence of breast carcinomas may be attributed to the presence of breast CSCs (BCSCs). BCSCs exist in mesenchymal-like or epithelial-like states and the transition between these states endows BCSCs with the capacity for tumor progression. The discovery of a feedback loop with galectins during epithelial-to-mesenchymal transition (EMT) prompted us to investigate its role in breast cancer stemness. Method To elucidate the role of Gal3 in BCSCs, we performed various in vitro and in vivo studies such as sphere-formation assays, Western blotting, flow cytometric apoptosis assays, and limited dilution xenotransplant models. Histological staining for Gal3 in tissue microarrays of breast cancer patients was performed to analyze the relationship of clinical outcome and Gal3 expression. Results Here, we show in a cohort of 87 node-positive breast cancer patients treated with doxorubicin-based chemotherapy that low Gal3 was associated with increased lymphovascular invasion and reduced overall survival. Analysis of in vitro BCSC models demonstrated that Gal3 knockdown by small hairpin RNA (shRNA) interference in epithelial-like mammary spheres leads to EMT, increased sphere-formation ability, drug-resistance, and heightened aldefluor activity. Furthermore, Gal3negative BCSCs were associated with enhanced tumorigenicity in orthotopic mouse models. Conclusions Thus, in at least some breast cancers, loss of Gal3 might be associated with EMT and cancer stemness-associated traits, predicts poor response to chemotherapy, and poor prognosis.