The E2f6 transcriptional repressor is an E2F-family member essential for the silencing of a group of meiosis-specific genes in somatic tissues. Although E2f6 has been shown to associate with both polycomb repressive complexes (PRC) and the methyltransferase Dnmt3b, the cross-talk between these repressive machineries during E2f6-mediated gene silencing has not been clearly demonstrated yet. In particular, it remains largely undetermined when and how E2f6 establishes repression of meiotic genes during embryonic development. We demonstrate here that the inactivation of a group of E2f6 targeted genes, including Stag3 and Smc1β, first occurs at the transition from mouse embryonic stem cells (ESCs) to epiblast stem cells (EpiSCs), which represent pre- and post-implantation stages, respectively. This process was accompanied by de novo methylation of their promoters. Of interest, despite a clear difference in DNA methylation status, E2f6 was similarly bound to the proximal promoter regions both in ESCs and EpiSCs. Neither E2f6 nor Dnmt3b overexpression in ESCs decreased meiotic gene expression or increased DNA methylation, indicating that additional factors are required for E2f6-mediated repression during the transition. When the SET domain of Ezh2, a core subunit of the PRC2 complex, was deleted, however, repression of Stag3 and Smc1β during embryoid body differentiation was largely impaired, indicating that the event required the enzymatic activity of Ezh2. In addition, repression of Stag3 and Smc1β occurred in the absence of Dnmt3b. The data presented here suggest a primary role of PRC2 in E2f6-mediated gene silencing of the meiotic genes.