It has been recently shown that endothelial platelet endothelial cell adhesion molecule‐1 (PECAM‐1) expression is pro‐atherogenic. PECAM‐1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane‐bound G proteins Gα_q/11 also mediate flow‐induced responses. Here, we investigated whether PECAM‐1 and Gα_q/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co‐localization of Gα_q/11 and PECAM‐1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gα_q/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM‐1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gα_q/11–PECAM‐1 complex within 30 s and a partial relocalization of the Gα_q/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow‐induced Gα_q/11–PECAM‐1 dissociation. These results allow us to conclude that Gα_q/11–PECAM‐1 forms a mechanosensitive complex and its localization suggests the Gα_q/11–PECAM‐1 complex is a critical mediator of vascular diseases.