Background and Aims: Activation of the cAMP/Epac signalling stabilises endothelial barrier function. cAMP/Epac also activates PI3K/Akt and MEK/ERK signalling in diverse cell types, but the impact of this activation on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed. Methods: Endothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring albumin flux. A modified cAMP analogue 8-pCPT-2′-O-Me-cAMP was used to specifically activate cAMP/Epac signalling. Activation of PI3K, Akt, MEK, and ERK was measured by Western blotting. Caspase activity was determined by Caspase-Glo kit. qPCR was used to quantify the expression level of PI3K isoforms. Cytoskeletal proteins and cell-cell junctions were visualised by immunohistochemistry using phalloidin TRITC and antibody against VE-cadherin, respectively. Endothelial cell proliferation was determined by expression of Ki67. Results: The Epac agonist reduced basal and significantly attenuated thrombin-induced endothelial hyperpermeability (n=5; p<0.05 for all further parameters); this was accompanied by an activation of PI3K/Akt and MEK/ERK signalling. Quantitative (q)PCR demonstrated that HUVECs express PI3Kα, PI3Kβ, and PI3Kγ but not PI3Kδ isoforms. Western blotting confirmed that the Epac agonist preferentially activates PI3Kα and PI3Kβ isoforms (n=3). Inhibition of the MEK/ERK using U0126 but not the PI3K/Akt pathway by Akt inhibitor VIII, potentiated the endothelial barrier-protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of the Epac agonist induced reorganisation of the actin cytoskeleton to the cell periphery, reduced stress fibre formation, and enhanced VE-cadherin localisation at cell-cell junctions. Moreover, the Epac agonist promoted endothelial cell (EC) survival but not proliferation via reduction in activities of pro-apoptotic caspases (caspase 3 and 7) in a PI3K/Akt and MEK/ERK signalling-dependent manner as determined by respective selective pharmacological inhibitors. Conclusion: Our data demonstrate that stimulation of the cAMP/Epac axis simultaneously activates PI3K/Akt and MEK/ERK pathways, which govern the pro-survival effects of Epac signalling on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier-stabilising and barrier-protective effects of cAMP/Epac activation.