T lymphocytes are exposed to hypoxia during their development and when they migrate to hypoxic pathological sites. Although it has been shown that hypoxia inhibits Kv1.3 channels and proliferation in human T cells, the mechanisms by which hypoxia regulates T cell activation are not fully understood. Herein we test the hypothesis that hypoxic inhibition of Kv1.3 channels induces membrane depolarization, thus modulating the increase in cytoplasmic Ca2+ that occurs during activation. Hypoxia causes membrane depolarization in human CD3+ T cells, as measured by fluorescence-activated cell sorting (FACS) with the voltage-sensitive dye DiBAC4(3). Similar depolarization is produced by the selective Kv1.3 channel blockers ShK-Dap22 and margatoxin. Furthermore, pre-exposure to such blockers prevents any further depolarization by hypoxia. Since membrane depolarization is unfavourable to the influx of Ca2+ through the CRAC channels (necessary to drive many events in T cell activation such as cytokine production and proliferation), the effect of hypoxia on T cell receptor-mediated increase in cytoplasmic Ca2+ was determined using fura-2. Hypoxia depresses the increase in Ca2+ induced by anti-CD3/CD28 antibodies in approximately 50% of lymphocytes. In the remaining cells, hypoxia either did not elicit any change or produced a small increase in cytoplasmic Ca2+. Similar effects were observed in resting and pre-activated CD3+ cells and were mimicked by ShK-Dap22. These effects appear to be mediated solely by Kv1.3 channels, as we find no influence of hypoxia on IKCa1 and CRAC channels. Our findings indicate that hypoxia modulates Ca2+ homeostasis in T cells via Kv1.3 channel inhibition and membrane depolarization.