Cancer stem cells (CSCs) are involved in cancer relapse and metastasis. These cells possess the ability to self-renew and differentiate into non-tumorigenic cell progeny. Additionally, the epithelial-mesenchymal transition (EMT) may either enhance or decrease the CSC population in a cell type-specific manner. We have demonstrated that GPC3 expression reverses the EMT undergone by breast cancer cells and modulates several signaling pathways, including Wnt/β-Catenin, which is involved in CSC regulation. Given the relevance of CSCs in metastasis and the roles of EMT and GPC3 in this process, we aimed to investigate the relationship between GPC3 and breast CSCs. We evaluated the mammosphere-forming ability of breast cancer cells with genetically modified GPC3 expression. We found that MDA-MB231-vector control cells form small clusters, whereas MDA-MB231-GPC3 cells developed large concentrically arranged mammospheres that were more numerous and had an average diameter about 50% larger than those of MDA-MB231-vector ones. On the other hand, the GPC3 silencing in MCF-7 cells did not affect morphology or size but resulted in a 3 to 4-fold reduction in mammosphere formation compared to control MCF-7 sh scramble cells. qPCR assays revealed that the pluripotency markers NANOG and OCT4 were poorly expressed in cell monolayers, independently of GPC3 presence. However, in silico studies showed the upregulation of stem markers such as SOX, ALDH1A1, and KLF4 in human mammary tumors classified as “high GPC3” (FDR0.5). Gene enrichment analysis using the TCGA database revealed that some signatures involved in stem cell maintenance and proliferation were positively regulated in the same group of patients (like GOBP_SOMATIC_STEM_CELL_POPULATION_MAINTENANCE, FDR<0.05, NES=1.51; GOBP_REGULATION_OF_STEM_CELL_PROLIFERATION, FDR<0.05, NES=1.39). In summary, our results highlight the role of GPC3 in CSC regulation and open a new research line to explore these find