ARTICLE ABSTRACTGlycoprotein nmb (GPNMB) is a type I transmembrane protein that contributes to the initiation and malignant progression of breast cancer through induction of epithelial–mesenchymal transition (EMT). Although it is known that EMT is associated with not only cancer invasion but also acquisition of cancer stem cell (CSC) properties, the function of GPNMB in this acquisition of CSC properties has yet to be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine the correlation between GPNMB and CSC properties in breast cancer cells. Three-dimensional sphere cultures induced higher expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than the 2D monolayer cultures. Three-dimensional culture also induced cell surface expression of GPNMB on limited numbers of cells in the spheres, whereas the 2D cultures did not. Therefore, we isolated cell surface-GPNMBhigh and -GPNMBlow cells from the spheres. Cell surface-GPNMBhigh cells expressed high levels of CSC genes and EMT-TF genes, had significantly higher sphere-forming frequencies than the cell surface-GPNMBlow cells, and showed no detectable levels of proliferation marker genes. Similar results were obtained from transplanted breast tumors. Furthermore, wild-type GPNMB, but not mutant GPNMB (YF), which lacks tumorigenic activity, induced CSC-like properties in breast epithelial cells. These findings suggest that GPNMB is exposed on the surface of dormant breast cancer cells and its activity contributes to the acquisition of stem cell–like properties.Significance: These findings suggest that cell surface expression of GPNMB could serve as a marker and promising therapeutic target of breast cancer cells with stem cell-like properties. Cancer Res; 78(22); 6424–35. ©2018 AACR.