Breast tumors are heterogeneous and composed of different sub-population of cells, each with dynamic roles that can change with stage, site and microenvironment. Cellular heterogeneity is in part due to cancer stem-like cells (CSC) that share properties with stem cells and associated with treatment-resistance. CSCs rewire metabolism to meet energy demands of increased growth and biosynthesis. O-GlcNAc transferase enzyme (OGT) uses UDP-GlcNAc as a substrate for adding O-GlcNAc moieties to nuclear and cytoplasmic proteins. OGT/O-GlcNAc levels are elevated in multiple cancers and reducing OGT in cancer cells blocks tumor growth. Here, we report that breast CSCs enriched in mammosphere cultures contain elevated OGT/O-GlcNAcylation. Inhibition of OGT genetically or pharmacologically reduced mammosphere forming efficiency, the CD44H/CD24L, NANOG+ and ALDH+ CSC population in breast cancer cells. Conversely, breast cancer cells over-expressing OGT increased mammosphere formation, CSC populations in-vitro and also increased tumor initiation and CSC frequency in-vivo. Furthermore, OGT regulates expression of a number of epithelial to mesenchymal transition (EMT) and cancer stem-like cell markers including CD44, NANOG, and c-Myc. In addition, we identify KLF8 as a novel regulator of breast cancer mammosphere formation and a critical target of OGT in regulating cancer stem-like cells. Implications: These findings demonstrate that OGT plays a key role in the regulation of breast CSCs in-vitro and tumor initiation in-vivo, in part, via regulation of KLF8 and thus inhibition of OGT may serve as a therapeutic strategy to regulate tumor-initiating activity.,Copyright ©2020, American Association for Cancer Research.