Abstract: Objective To investigate the effect of miR-26b on the proliferation， apoptosis and stemness of the breast cancer cells as well as the related mechanism. Methods The MCF-7 cell was cultured with serum free suspension culture in order to obtain the MCF-7 stem cells. Real time fluorescence quantitative PCR （qRT-PCR） was used to detect the miR-26b levels of MCF-7 and its stem cells. According to the experiment protocol， the MCF-7 stem cells were divided into 3 groups： control group， empty transfection group（cells transfected with con-mimics） and overexpression group（cells transfected with miR-26b mimics）. MTT assay was used to measure the proliferation ability at different times （24， 48， 72 and 96 h） after transfection. Flow cytometry was used to detect the level of apoptosis and the number of side population （SP） cells after transfection. The sphere rate after transfection was assessed by stem cells culture in vitro. The mRNA levels of Lin-28B， ALDH-1， BMI-1， OCT-4 and CD44 were detected by qRT-PCR after transfection. The verification of miR26b target genes of glycogen synthase kinase 3β （GSK-3β） were evaluated by biological information science and Western blotting analysis. Results The miR-26b level of MCF-7 stem cells was lower than that of MCF-7 cells （P＜0.05）. The transfection of miR-26b mimics could increase the miR26b level of MCF-7 stem cells. Compared with the control group， the proliferation inhibition rate and apoptosis rate were increased， but the number of SP cells and sphere rate were decreased in overexpression group （P＜0.05）. Transfection of miR-26b mimics could lead to lower levels of Lin-28B， BMI-1， ALDH-1， OCT-4 mRNA （P＜0.05）. Bioinformatics software predicted GSK-3β as the potential target gene of miR-26b. Transfection of miR-26b mimics could significantly reduce the expression of GSK-3β， and double luciferase reporter gene assay proved that miR-26b could predict the target position in the 3’UTR mRNA region of GSK-3β gene. Conclusion MiR-26b was lowlyexpressed in MCF-7 stem cells. MiR-26b can inhibit cell proliferation， induce apoptosis and negatively regulate the stemness of breast cancer cells， which may be related to the down-regulation of GSK-3β expression.