In endothelial cells, caveolin-1 (cav-1), the structural protein of caveolae, acts as scaffolding protein to cluster lipids and signaling molecules within caveolae and in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium derived hyperpolarizing factor (EDHF)-mediated relaxation are located in caveolae and/or regulated by the structural protein cav-1, such as potassium channels, calcium regulatory proteins and connexin 43 (Cx), one molecular component of gap-junctions. Comparing relaxation in vessels from cav-1 knock-out mice (cav-1 KO) and their wild type (WT) littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from cav-1 KO mice. The absence of cav-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably a decreased activity of Ca2+-permeable TRPV4 cation channels that participate in the NO- and EDHF-mediated relaxation. Moreover, morphological characterization of cav-1 KO and WT arteries showed less gap-junction in vessels from KO animals associated with a lower expression of connexins 37, 40, 43 and an altered myo-endothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with cav-1 in the caveolar compartment of the plasma membrane. In conclusion we demonstrated that expression of cav-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins that are both implicated at different steps in the EDHF-signaling pathway.