A prominent feature of sickle cell anemia is the presence of dehydrated red blood cells (RBCs) in circulation. Loss of potassium (K⁺), chloride (Cl⁻), and water from RBCs is thought to contribute to the production of these dehydrated cells. One main route of K⁺ loss in the RBC is the Gardos channel, a calcium (Ca²⁺)–activated K⁺ channel. Clotrimazole (CLT), an inhibitor of the Gardos channel, has been shown to reduce RBC dehydration in vitro and in vivo. We have developed a chemically novel compound, ICA-17043, that has greater potency and selectivity than CLT in inhibiting the Gardos channel. ICA-17043 blocked Ca²⁺-induced rubidium flux from human RBCs with an IC₅₀ value of 11 ± 2 nM (CLT IC₅₀ = 100 ± 12 nM) and inhibited RBC dehydration with an IC₅₀ of 30 ± 20 nM. In a transgenic mouse model of sickle cell disease (SAD), treatment with ICA-17043 (10 mg/kg orally, twice a day) for 21 days showed a marked and constant inhibition of the Gardos channel activity (with an average inhibition of 90% ± 27%, P < .005), an increase in RBC K⁺ content (from 392 ± 19.9 to 479.2 ± 40 mmol/kg hemoglobin [Hb], P < .005), a significant increase in hematocrit (Hct) (from 0.435 ± 0.007 to 0.509 ± 0.022 [43.5% ± 0.7% to 50.9% ± 2.2%], P< .005), a decrease in mean corpuscular hemoglobin concentration (MCHC) (from 340 ± 9.0 to 300 ± 15 g/L [34.0 ± 0.9 to 30 ± 1.5 g/dL], P < .05), and a left-shift in RBC density curves. These data indicate that ICA-17043 is a potent inhibitor of the Gardos channel and ameliorates RBC dehydration in the SAD mouse.