Background and Aims: Activation of the cAMP/Epac signalling stabilises endothelial cell (EC) barrier function. cAMP/Epac also activates PI3K/Akt and MEK/ERK signalling in ECs but their impact on EC barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated EC barrier stabilisation was analysed. Methods: EC barrier function was analysed in cultured human umbilical vein ECs (HUVECs) by measuring flux of albumin. 8-pCPT-2′-O-Me-cAMP (200 μM) was used to specifically activate cAMP/Epac signalling. Results: Activation of cAMP/Epac signalling reduced the basal and attenuated thrombininduced EC hyperpermeability. The reduction in permeability was accompanied by an activation of PI3Kα and PI3Kβ, MEK/ERK, and re-organisation of actin cytoskeleton and VE-cadherin at adherens junctions (AJs). However, it did not affect RhoA/Rock activity. Inhibition of PI3K/Akt signalling by isoform specific PI3K inhibitors or a specific Akt inhibitor (inhibitor viii; 5 μM) had no effect on Epac-mediated EC barrier stabilisation but abrogated Epac agonist-mediated EC proliferation. On the other hand, inhibition of MEK/ERK pathway by specific MEK inhibitors (U0126 and PD98059; 10 μM) potentiated the EC barrier stabilising effect of cAMP/Epac. Inhibition of MEK/ERK pathway had no effect on RhoA/Rock signalling but antagonised activation of contractile machinery, and enhanced Epac-mediated re-arrangement of actin and VE-cadherin at cell borders thus strengthening AJs. Conclusion: Epac agonist activates PI3Kα and PI3Kβ and MEK/ERK signalling. Both PI3K/Akt and MEK/ERK signalling play important role in Epac-mediated EC proliferation. Inhibition of MEK/ERK but not PI3K/Akt signalling potentiates EC barrier protective effect via inhibition of EC contractile machinery.