〔Abstract〕 Objective To clarify the regulatory role of miRNA-26 b(miR-26 b) in the pluripotency of mouse embryonic stem cells(mESCs). Methods Real-time quantitative PCR was used to detect the expression of miR-26 cluster precursors in mESCs and MEF.Real-time quantitative PCR was used to detect the expression of miR-26 b during the formation of embryoid body(EB).CCK-8 assay was used to detect the effect of miR-26 b on the proliferation ability of mESCs.Flow cytometry was used to detect the regulatory effect of miR-26 b on cell cycle and apoptosis of mESCs.Alkaline phosphatase staining kit was used to detect the regulatory effect of miR-26 b on ALP activity in mESCs.Real-time quantitative PCR was used to detect the effect of miR-26 b on the mRNA expression of pluripotent transcription factors(Oct4, Sox2 and Nanog) in mESCs.Western blot was used to detect the effects of miR-26 b on the protein expression of three transcription factors in mESCs.The morphology of mESCs after transfecting pcDNA-pre-miR-26 bin vitrowas observed. Results In mESCs, the expression of pre-miR-26 b was highest among all members in miR-26 clusters. All miR-26 precursors were up-regulated in differentiated mouse embryonic fibroblasts(MEF), pre-miR-26 b showed the most significant increase among the three members(about 10-fold increase).Mature miR-26 b was significantly up-regulated from day 2 to day 6 during embryonic body(EB) formation, and the expression level of mature miR-26 b in MEF was significantly higher than that in mESCs.MiR-26 b overexpression repressed mESCs proliferation and arrested cell cycle in G1 phase. MiR-26 b did not affect mESCs apoptosis.Alkaline phosphatase staining showed that miR-26 b decreased the activity of ALP in mESCs.MiR-26 b overexpressing mESCs reduced expression of pluripotency markers(Oct4, Sox2 and Nanog).The effect of miR-26 b on mESCs differentiation was studied using anin vitrodifferentiation model. Differentiation was observed as early as day 4 of miR-26 b overexpression. Conclusion This study confirms the regulatory effects of miR-26 b on mESCs pluripotency.