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Background and Purpose The molecular identity of calcium-activated chloride channels ( Ca CCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 ( Ano1, also known as TMEM16 A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells ( CVECs). Experimental Approach Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. Key Results Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for Ca CCs. The density of ICl( Ca) detected in CVECs was significantly inhibited by T16 Ainh- A01, an Ano1 inhibitor, and a pore-targeting, specific anti- Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl( Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl( Ca) was inhibited by T16 Ainh- A01 or anti- Ano1 antibody. Hypoxia also increased the current density of ICl( Ca) in Ano1 gene knockdown CVECs. Conclusions and Implications Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl( Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca2+. Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage. [ABSTRACT FROM AUTHOR]