The POU5F1 gene encodes one of the "core" transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5' regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere with POU5F1 expression and function in in vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends on de novo methylation of its promoter. However, little is known about the epigenetic regulation of POU5F1 expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of the POU5F1 5'upstream region, relative to its regulated expression in different compartments of the blastocyst over a two-day period of development. We evidenced progressive methylation of the 5' regulatory region and the first exon accompanying differentiation, and the gradual repression of POU5F1. Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naive pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.