The POU5F1 gene encodes one of the "core" transcription factors necessary to maintain pluripotency but very few data are available concerning the precise epigenetic regulation of its expression in early embryo. We analysed the progressive modifications of DNA methylation of POU5F1 upstream region in the different compartments of the developing blastocyst. Therefore we used the rabbit embryo as a model for most mammalian blastocysts, where contrarily to the mouse, epiblast differentiates in a plane embryonic disk at the surface of the conceptus and in direct contact with maternal environment. Embryos were micro dissected at Day4,5, and6. POU5F1 expression was quantified by RTqPCR. Methylation profile of four regions encompassing conserved cis-elements (Kobolak et al. 2009 BMC Mol. Biol.10 88.) was determined by bisulfite treatment, cloning and sequencing. POU5F1 is highly expressed in Day4 trophectoderm but significantly enriched in the inner cell mass. At Day5, the POU5F1 relative expression decreased but the difference between embryonic and trophectoderm compartments tends to increase. At Day6, POU5F1 transcripts were restricted to the epiblast, with a very reduced expression in the hypoblast. Noticeably POU5F1 expression progressively decreases in the trophectoderm/trophoblast compartment over the Day4-Day6 period. The four regions were hypomethylated in pluripotent Inner Cell Mass and epiblast. Differences in DNA methylation between pluripotent and differentiated layers occurred as early as Day4 and progressively increased. A principal component analysis segregated pluripotent and highly differentiated samples and identified the CpGs mostly involved in this separation. Interestingly while differentiation-related changes in methylation occurred in the four analyzed regions, theyare more pronounced in the region encompassing the proximal enhancer 1A.