Mechanochemical signal transduction occurs when mechanical forces, such as fluid shear stress, are converted into biochemical responses within the cell. The molecular mechanisms by which endothelial cells (ECs) sense/transduce shear stress into biological signals, including the nature of the mechanosensor, are still unclear. G proteins and G protein-coupled receptors (GPCRs) have been postulated independently to mediate mechanotransduction. In this study, we used in situ proximity ligation assay (PLA) to investigate the role of a specific GPCR/Gα_q/11 pair in EC shear stress-induced mechanotransduction. We demonstrated that S1P stimulation causes a rapid dissociation at 0.5 min of Gα_q/11 from its receptor, S1P₃, followed by an increased association within 2 min of GRK2 and β-arrestin 1/2 with S1P₃ in human coronary artery ECs, which are consistent with GPCR/Gα_q/11 activation and receptor desensitization/internalization. The G protein activator, AlF₄^-, resulted in increased dissociation of Gα_q/11 from S1P₃, but no increase in association between S1P₃ and either GRK2 or β-arrestin 1/2. The G protein inhibitor, GDP-β-S, and the S1P₃ antagonist, VPC23019, both prevented S1P-induced activation. Shear stress also caused the rapid activation within 7 sec of S1P₃/Gα_q/11. There were no increased associations between S1P₃ and GRK2 or S1P₃ and β-arrestin 1/2 until 5 min. GDP-β-S, but not VPC23019, prevented dissociation of Gα_q/11 from S1P₃ in response to shear stress. Shear stress did not induce rapid dephosphorylation of β-arrestin 1 nor rapid internalization of S1P3, indicating no GPCR activation. These findings suggest that Gα_q/11 participates in the sensing/transducing of shear stress independent of GPCR activation in ECs.