Endothelial responses to haemodynamic forces associated with blood flow play a pivotal role in the acute regulation of vascular tone. Regulation of endothelial intracellular Ca2+ level plays a key role in controlling vascular contraction and relaxation. It is not yet understood how blood vessels sense mechanical forces, but it involves at least one mechanosensitive molecule at the cell surface that detects the force and transduces it into a biochemical signal. Mechanosensitive Ca2+ permeable ion channels are an important class of proteins that respond to mechanical stimuli and are expressed on the endothelial membrane. There is a growing appreciation that local increases in intracellular Ca2+ level ([Ca2+]i) are important for the regulation of vascular function. Transient receptor potential vanilloid 4 (TRPV4), a non-selective cation channel, is a candidate mechanoreceptor that is widely expressed in the endothelium. Ca2+ influx through TRPV4 is an important source of local Ca2+ increase in the endothelium and it has been shown that the cooperative opening of as few as three clustered TRPV4 channels, per endothelial cell causes maximal vasodilation through the activation of intermediate- and small- conductance, Ca2+ sensitive potassium channels (Sonkusare et al., 2014). Using a combination of super resolution microscopy, cell surface biotinylation assay and Ca2+ imaging, we have shown that TRPV4 channels form small clusters at the endothelial cell membrane that are in complex with component of adherens junctions such as VE-cadherin and β-catenin. Shear stress stimulation increased the density of TRPV4 channels at the cell membrane and also increased the density and size of TRPV4 clusters. Shear stress stimulation resulted in trafficking of TRPV4 channels from the basolateral membrane to the basal membrane, and reduced the density of TRPV4 channels in complexes with VE-cadherin and β-catenin. TRPV4 trafficking to the cell membrane was regulated by AKT, β1 integrin and Rac1 signalling pathways but was independent of PI3K.