The purpose of the study was to detect the effect and possible mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) on the in vitro and in vivo growth of stem cells isolated from primary human breast cancer cells and cell lines MDA-MB-231 and MCF-7. Primary human breast cancer cells and MDA-MB-231 and MCF-7 cells were sorted in vitro using flow cytometry, and the ESA+, CD44+, CD24-/low cells were isolated as breast cancer stem cells (CSCs). The inhibitory effect of hUCMSCs on CSCs was examined using the Cell Counting Kit-8 cell proliferation and soft agar colony formation assay. In vivo tumor inhibition was studied using a severe combined immunodeficient xenograft mouse model transplanted with MDA-MB-231 breast CSCs. The expression of phosphoinositide 3-kinase (PI3K) and AKT was examined in the xenograft tumors using immunohistochemistry. The number of colonies formed by breast CSCs co-cultured with hUCMSCs at the bottom of soft agar was significantly lower than those formed by the control group (P < 0.01). Compared with the control group, the CSCs co-cultured with hUCMSCs showed a higher number of cells in the G2-M phase (P < 0.05) and an increased number of apoptotic cells (P < 0.01). The mice in the medium- and high-concentration hUCMSC treatment groups exhibited clearly reduced tumor volume and tumor weight, compared with the control group (P < 0.01). Compared with the saline group, the xenograft tumor tissues from the mice treated with different concentrations of hUCMSCs showed significantly reduced levels of PI3K and AKT proteins (P < 0.001). In conclusion, hUCMSC significantly inhibited the growth of breast CSCs in vitro and in vivo. The underlying mechanism is likely related to cell cycle arrest, induction of tumor cell apoptosis, and suppressed activities of PI3K and AKT protein kinases.