Background and Aims: Elevated intracellular cAMP levels stabilise endothelial cell (EC) barrier function via PKA-dependent modulation of the activities of Rho GTPases. In the present study the role of Rho GTPases, RhoA and Rac1, in cAMP/PKA-mediated in vitro angiogenesis was investigated. Methods: The study was carried out on cultured human umbilical vein ECs (HUVECs). cAMP analogue N6-Benzoyl-cAMP (50 µM) and forskolin (FSK; 10 µM) were used to activate PKA or adenylyl cyclase (AC), respectively. In vitro angiogenesis was analysed by 3-D spheroid assay and matrigel tube formation assay. RhoA activity was modified by lentiviral-mediated over expression of dominant negative and constitutive active forms of RhoA. Results: Specific activation of either PKA or AC induced EC proliferation, migration, tube formation, and 3-D spheroids and potentiated the VEGF-induced in vitro angiogenesis. Activation of cAMP/PKA-signalling induced phosphorylation of VEGFR2 and Akt, an inhibition of RhoA/Rock pathway, and activation of Rac1. Pharmacological activation of RhoA but not Rac1 abrogated PKA-induced VEGF receptor phosphorylation, tube formation and 3-D spheroids. Lentiviral mediated over-expression of constitutive active form of RhoA abrogated PKA- but not VEGF-induced VEGFR2 and Akt phosphorylation and angiogenesis. Moreover, pharmacological inhibition of Akt also abrogated PKA-mediated VEGFR2 phosphorylation and angiogenesis. On the other hand over expression of dominant negative RhoA or inhibition of Rock (fasudil) promoted basal and potentiated PKA-mediated phosphorylation of Akt and VEGFR2 and in vitro angiogenesis. Conclusion: We describe for the first time a novel interplay between PKA, Rock, Akt, and VEGFR2 signalling and angiogenesis. Inhibition of RhoA/Rock plays an important role in mediating PKA-induced angiogenesis and activation of RhoA/Rock signalling and can be an important target to intervene therapeutic angiogenesis.