Endothelial cells undergo a rapid cell–cell junction inclination following exposure to atheroprotective unidirectional flow. In contrast, atherosclerotic lesions correlate with a heterogeneous distribution of the junctional wall inclination in cells exposed to time-varying, reversing, and oscillatory flow as well as to low mean shear stress. However, the underlying biochemical events by which endothelial cells distinctively respond to unidirectional versus flow reversal remain unclear. Here, we show that the subcellular distribution of flow-induced Akt-1 phosphorylation in endothelial cells lining the mouse aorta varies depending on local hemodynamics. Activated Akt-1 accumulated in perinuclear areas of cells in regions predisposed to disturbed flow but were localized at the cell–cell junction in regions of high unidirectional laminar shear stress. In flow-adapted human endothelial cells, reversal in flow direction was associated within minutes with a subcellular concentration of phosphorylated Akt-1 at the upstream edge of cells. Interestingly, oscillatory flow (with a zero mean shear stress) failed to activate Akt-1, whereas a unidirectional pulsatile flow of similar amplitude induced an increase in Akt-1 phosphorylation. Finally, silencing of the G protein αq/11 subunit abrogated both flow-induced Akt-1 and GSK-3β activation. Together, these results characterize the existence of a Gαq/11-mediated Akt-1 signaling pathway that is dynamically responsive to flow direction, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to flow reversal. J. Cell. Biochem. 115: 121–129, 2014. © 2013 Wiley Periodicals, Inc.