Stimulation of proteinase-activated receptor 2 (PAR2) on endothelial cells (ECs) leads to an increase in intracellular Ca2+ stimulating endothelium-derived hyperpolarization (EDH), which spreads to adjacent vascular smooth muscle (VSM) causing vasodilation. In the resistance vasculature, EDH can also rapidly spread to neighbouring ECs to trigger VSM relaxation along the length of the artery in a process termed conducted vasodilation. Previously, mouse models of diabetes mellitus have been reported to exhibit diminished EC Ca2+signalling and EDH. However, the effect of chronic hyperglycaemia, the hallmark of diabetes mellitus, on conducted dilation has not been characterized. Small mesenteric arteries were isolated from healthy and diabetic db/db mice, which were used as a model of chronic hyperglycaemia. Endothelium-dependent vasodilation via the Gq/11-coupled proteinase-activated receptor 2 (PAR2) was stimulated with the selective agonist SLIGRL. The Ca2+-sensitive fluorescent indicator fluo-8 reported changes in endothelial cell (EC) [Ca2+]i, and triple-cannulated bifurcating mesenteric arteries were used to study conducted vasodilation. Localized intraluminal perfusion of SLIGRL into the bifurcation of triple-cannulated arteries from WT mice induced conducted vasodilation 2.5mm away from the initial site of local vasodilation (Figure 1). This conducted response was reduced during acute exposure to a high glucose (40mM) or hyperosmolar (29mM mannitol) solution. Furthermore, arteries isolated from db/db mice also exhibited diminished conducted vasodilation. However, chronic hyperglycaemia did not affect either EC Ca2+mobilization or EDH-dependent vasodilation stimulated by SLIGRL. The current investigation demonstrates for the first time that the spread of a hyperpolarizing current along the endothelium of the resistance vasculature is attenuated in a mouse model of chronic hyperglycaemia. In addition, diminished conducted vasodilation was also observed during acute exposure to a hypertonic solution containing glucose or mannitol. Our findings reiterate the importance of studying the effects of hyperglycaemia in the vasculature, and provide the basis for further studies regarding the modulation of junctional proteins involved in cell to cell communication by diseases such as diabetes.