Objective ·To explore the effect of double homeobox (DUX) protein on the differentiation potential of mouse embryonic stem cells (mESCs) into extraembryonic endoderm (XEN) and the possible mechanism of its action. Methods ·Overexpression of DUX cell lines in mESCs was achieved by using a lentiviral system. The proportion of 2-cell-like cells (2CLCs) before and after DUX overexpression was detected by flow cytometry, and the expression of 2-cell stage-specific genes, Dux, zinc finger and SCAN domain containing 4c (Zscan4c), zinc finger protein 352 (Zfp352) and murine endogenous retrovirus-L polymerase (MERVL-pol), were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). RT-qPCR assay was used to detect the expression of pluripotency factors, nanog homeobox (Nanog), kruppel-like transcription factor 4 (Klf4), sex determining region Y-box 2 (Sox2), and octamer-binding transcription factor 4 (Oct4), in pluripotent state, as well as the expression of signature genes for different germ layers in the differentiated state [endodermal: GATA binding protein 4 (Gata4), GATA binding protein 6 (Gata6), and sex determining region Y-box 17 (Sox17); ectodermal: Nestin and tubulin beta 3 class Ⅲ (Tubb3); mesodermal: heart and neural crest derivatives expressed 1 (Hand1), myogenic differentiation 1 (Myod1), and kinase insert domain protein receptor (Flk1)]. Public RNA sequencing (RNA-seq) data were mined to further clarify the effect of DUX on the differentiation of mESCs into extraembryonic endoderm. Functional and pathway enrichment analyses of differentially expressed genes were performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) to identify the signaling pathways regulated by DUX. Additionally, an in-depth analysis of existing chromatin immunoprecipitation sequencing (ChIP-seq) data was conducted to explore the potential target genes of DUX. Results ·Molecular biology experiments showed that overexpression of DUX could effectively maintain the pluripotency of mESCs, which was consistent with the analysis of public RNA-seq data. Differential gene analysis revealed that endodermal genes were specifically upregulated. After differentiation assay of mESCs, RT-qPCR assay experiments showed that mRNA expression of the XEN marker genes (Gata4, Gata6, Sox17) was significantly upregulated (P<0.001). In contrast, there was no specific change in mesodermal and ectodermal genes. GSEA enrichment analysis indicated that DUX might activate the retinoid metabolism signaling pathway, and the analysis of the ChIP-seq data further revealed the presence of a large number of known retinoic acid receptor motif in DUX-bound peaks, which could activate downstream target genes related to the development of the XEN. Conclusion ·DUX has a strong correlation with the retinoic acid signaling pathway and it is predicted to activate the retinoic acid signaling pathway, which could promote the tendency of mESCs toward XEN differentiation.