Endothelial cell dysfunction is a major cause of vascular disease that can be propagated by multiple pathological conditions, although the mechanisms of endothelial cell dysfunction are not fully understood. We have recently found that the activity of the store-operated Ca2+ channel molecules, STIM/ORAI, was enhanced by high glucose in vascular endothelial cells, which causes abnormal Ca2+ homeostasis and endothelial dysfunction under hyperglycaemia [1]. Since hyperglycaemia is usually accompanied by hyperosmolarity in diabetes, particularly for the critical condition called Hyperglycaemic Hyperosmolar State (HHS), we aimed to study the effect of hyperosmolarity on the expression and activity of STIM/ORAI channels in vascular endothelial cells. Using Fura-PE3 AM Ca2+ dye, we found that store-operated Ca2+ entry (SOCE) evoked by thapsigargin was inhibited in endothelial cells (EA.hy926) by the pre-treatment with hyperosmotic conditions via addition of 30, 60 and 90 mM mannitol in normal bath solution . The inhibition of SOCE by hyperosmolarity was also seen in the HEK293 cells overexpressing EYFP-STIM1/mCherry-ORAI1, EYFP-STIM1/mCherry-ORAI2, and EYFP-STIM1/mCherry-ORAI3 channels. The currents for ORAI1, ORAI2 and ORAI3 were examined by whole cell patch-clamp, and the thapsigargin-evoked currents of ORAI1 and ORAI2, and 2-APB-induced ORAI3 were inhibited by hyperosmolar condition (19.5 mM mannitol) in the HEK293 overexpressing STIM1/ORAI1-3 channels. The mRNA and protein expression of ORAI1-3/ STIM1-2 was detected by real-time PCR and Western blotting, respectively, and their expression levels were down-regulated by hyperosmotic conditions. Endothelial cell (EA.hy926) migration was significantly inhibited by hyperosmolarity after incubation with 19.5 mM mannitol or 19.5 mM sucrose for 24 hrs using a scratch wound healing assay , however hyperosmolarity caused by addition of 19.5 mM mannitol and 19.5 mM sucrose had no effect on endothelial cell proliferation measured by WST-1 assay. The cell volume was significantly reduced by hyperosmotic conditions (30, 60 and 90 mM mannitol), but such cell volume change could be unrelated to the expression of ORAI/STIM channels, because there was no significant difference between the non-transfected cells and the ORAI/STIM transfected cells. Our findings suggest that SOCE is regulated by hyperosmolarity in vascular endothelial cells and ORAI/STIM channels contribute the cellular processes. The relevance of SOCE to osmotic stress could provide a new insight to the pathophysiology of endothelial dysfunction in diabetes.