--- - |2+ Key points It has been postulated that increased blood flow‐associated shear stress on endothelial cells is an underlying mechanism by which physical activity enhances insulin‐stimulated vasodilatation. This report provides evidence supporting the hypothesis that increased shear stress exerts insulin‐sensitizing effects in the vasculature and this evidence is based on experiments in vitro in endothelial cells, ex vivo in isolated arterioles and in vivo in humans. Given the recognition that vascular insulin signalling, and associated enhanced microvascular perfusion, contributes to glycaemic control and maintenance of vascular health, strategies that stimulate an increase in limb blood flow and shear stress have the potential to have profound metabolic and vascular benefits mediated by improvements in endothelial insulin sensitivity. Abstract The vasodilator actions of insulin contribute to glucose uptake by skeletal muscle, and previous studies have demonstrated that acute and chronic physical activity improves insulin‐stimulated vasodilatation and glucose uptake. Because this effect of exercise primarily manifests in vascular beds highly perfused during exercise, it has been postulated that increased blood flow‐associated shear stress on endothelial cells is an underlying mechanism by which physical activity enhances insulin‐stimulated vasodilatation. Accordingly, herein we tested the hypothesis that increased shear stress, in the absence of muscle contraction, can acutely render the vascular endothelium more insulin‐responsive. To test this hypothesis, complementary experiments were conducted using (1) cultured endothelial cells, (2) isolated and pressurized skeletal muscle arterioles from swine, and (3) humans. In cultured endothelial cells, 1 h of increased shear stress from 3 to 20 dynes cm−2 caused a significant shift in insulin signalling characterized by greater activation of eNOS relative to MAPK. Similarly, isolated arterioles exposed to 1 h of intraluminal shear stress (20 dynes cm−2) subsequently exhibited greater insulin‐induced vasodilatation compared to arterioles kept under no‐flow conditions. Finally, we found in humans that increased leg blood flow induced by unilateral limb heating for 1 h subsequently augmented insulin‐stimulated popliteal artery blood flow and muscle perfusion. In aggregate, these findings across models (cells, isolated arterioles and humans) support the hypothesis that elevated shear stress causes the vascular endothelium to become more insulin‐responsive and thus are consistent with the notion that shear stress may be a principal mechanism by which physical activity enhances insulin‐stimulated vasodilatation. - 'The Journal of Physiology, EarlyView. '