To learn about the role of RNA-binding protein 25(RBM25) in cell-fate regulation of mouse embryonic stem cells(mESCs), this study established a stable Rbm25-knockdown mESC line using shRNA and systematically evaluated its function in pluripotency maintenance and 2-cell-like(2C-like) conversion by integrating public RNA-seq/CUT&Tag datasets, bioinformatic analyses, qRT-PCR, Western blot, alkaline phosphatase staining, a 2C::tdTomato reporter system, CUT&Tag-qPCR, and dual-luciferase assays. The results showed that Rbm25 depletion disrupted colony morphology and alkaline phosphatase activity, reduced Oct4GiP reporter positivity, and markedly decreased the transcriptional and protein expression of the core pluripotency genes Oct4, Sox2, Klf4, and Nanog. And, differentiation-associated genes, including Gata3, Gata4, Gata6, Foxa2, Cdx2, and Wnt9a, were up-regulated, whereas 2C-specific genes such as MERVL, Zscan4, Tcstv1, Tcstv3, Zfp352, and Sp110 were robustly activated, accompanied by a significant increase in the proportion of 2C-like cells. RBM25 was enriched at the promoter regions of key PRC1.6 subunit genes, including Rnf2, Pcgf6, Max, and Mga, and enhanced their promoter activities. Loss of Rbm25 reduced the expression of these PRC1.6 components, suggesting that repression of the 2C transcription was weakened. These findings identify RBM25 as an essential regulator of mESC pluripotency and a critical barrier to the 2C-like state transition, acting in part by transcriptionally activating core components of the PRC1.6 complex.