Endothelial cells, which line the artery walls, respond to fluid flow stimulation through activation of signalling pathways. Smad2, a signalling molecule and transcription factor typically associated with TGF-β signalling, is preferentially phosphorylated in the linker region (L- pSmad2) and localized to the nucleus in a flow-dependent manner in endothelial cells. Here, we show that L-pSmad2 expression is higher in the high shear stress region of the thoracic aorta than in the lower shear stress region of the lesser curvature of mice. L-pSmad2 was not affected by flow pulsatility in human endothelial cells and did not vary in disturbed flow versus laminar regions in carotid arteries of surgically modified mice. Smad2 siRNA treatment of human endothelial cells resulted in decreased Smad2 and increased protein levels of Akt under flow. These findings represent novel contributions to the knowledge of fluid flow-induced Smad2 signalling and its role in endothelial health.