Objective To investigate the effects of salinomycin on the cell proliferation and epithelial-mesenchymal transition (EMT) of MCF-7 mammosphere (MCF-7 MS). Methods Breast cancer MCF-7 cells were cultured in suspension in serum-free medium to obtain MCF-7 MS. The cell viability of MCF-7 MS cells treated with serial concentrations of 0, 10, 30, 100, 300, 1 000, 3 000 and 10 000 nmol/L of salinomycin for 24 hours were detected by CCK-8 assay. The half maximal inhibitory concentration (IC50) was calculated. Western blot analysis was performed to detect the expression levels of E-cadherin and Snail in MCF-7 MS cells treated with 30 nmol/L and 60 nmol/L salinomycin. The same capacity of DMSO was added to MCF-7 MS as control group. The xenograft tumors from MCF-7 MS transplant mice were divided into control group (the same capacity of normal saline) and salinomycin group (5 mg/kg salinomycin), then the expressions of E-cadherin and Snail were dectected by immunohistochemical staining. Results With the increased concentration of salinomycin, the cell survival rate of MCF-7 MS cells decreased (P<0.05). The IC50 after 24 h-treatment was 989 nmol/L. Both 30 and 60 nmol/L of salinomycin increased the expression of E-cadherin and decreased the expression of Snail compared with control group. In addition, 60 nmol/L treatment group showed more significant effect (P<0.05). In xenograft tumors from MCF-7 MS transplant mice, the expression of Snail decreased, and E-cadherin increased in salinomycin treatment group compared with control group (P<0.01). Conclusion Salinomycin can inhibit the cell proliferation and EMT in MCF-7 MS cells, which is a potential drug to target cancer stem cells.