The Ward group have previously demonstrated that E-cadherin regulates the naïve pluripotent state of mouse Embryonic Stem (mES) cells via activation of the LIF/STAT3 pathway. Loss of E-cadherin in mES cells leads to a 'primed' pluripotent phenotype characterized by LIF-independent self-renewal through ACTIVIN/NODAL signalling, similar to mouse Epiblast Stem (EpiS) and human (h)ES cells. However, the exact mechanism of E-cadherin's regulation of pluripotency in both mouse and human ES cells has remained elusive. In this study we describe a novel network modelling approach of transcriptomic analysis using gene expression changes shared between three independently derived E-cadherin negative cell lines (collectively termed ESDeltaEcad cells). Our models predicted regulation of pluripotency pathways via the co-activator protein EP300. We show that E-cadherin regulates the cell localisation and binding of EP300 to NANOG-OCT4-SOX2 DNA clusters. EP300 also functions to phosphorylate SMAD2 in the absence of E-cadherin and to inhibit pluripotency-associated transcripts in Ecad-/- mES cells. Using comparative network analysis of E-cadherin antagonism in mouse and human transcriptomes we have identified that E-cadherin plays a major role in regulating the transcriptional circuitry between naive and primed ES cell states. We show that this is likely to be mediated at least partly by positive activation of NCOA3 and ESRRB. Differential transcript expression in Ecad-/- mES cells correlates with loss of ESRRB protein expression and altered cellular localisation of NCOA3, both regulators of naive pluripotency. We have gone on to show through peptide antagonism that while E-cadherin is an important upstream regulator of genes critical in defining the naïve pluripotent state, this action is not necessarily dependent on the loss of cell-cell contact associated with E-cadherin inhibition. We present the first data that E-cadherin regulates naïve mES cell pluripotency by regulation of EP300, ESRRB and NCOA3 activity. Furthermore we have shown E-cadherin to inhibit the naïve transcriptional phenotype in hES cells. Peptides that target E-cadherin also provide a valuable tool for controlling the transition between naive and pluripotent states in mouse and human ES cells.