Renal iron deposition correlates with elevated plasma ET-1 in patients and mouse model of sickle cell disease (SCD). Additionally, excessive renal iron accumulation in sickle cell mice (HbSS) is ameliorated by ETA receptor antagonism. Thus, we hypothesized that ET-1 via ETA receptor activity leads to dysfunctional renal iron trafficking, promoting iron accumulation in the kidney. We conducted time- and concentration-dependent in vitro studies on mouse proximal tubule (PT) cells and observed that ET-1/ ETA signaling promotes cellular iron uptake (by increasing transferrin receptor 1 expression) and inhibits cellular iron removal pathways (65% reduction of ferroportin-1 (FPN-1) and doubled hepcidin (Hamp) expression). In vivo studies demonstrated that altered expression of iron transporters in PT of male HbSS mice was consistent with iron accumulation phenotype. ETA receptor blockade attenuated expression of the cellular iron uptake mediator, DMT-1, and also decreased renal iron accumulation (1.43±0.06 vs. 2.62±0.12; p=0.097) by preserving FPN-1, reducing Hamp expression, and increasing urinary iron excretion (10.9±1.0 vs 6.4±1.2 μg/24h; p=0.010). To determine the long-term effect of ET-1 on chronic renal iron accumulation, we generated HbSS mice lacking ET-1 in vascular endothelial cells mice (HbSS VEET KO; using Tie-2 Cre). There were no differences in anemia status between 20 weeks old male HbSS VEET KO and flox mice. However, plasma iron concentration (117.3±4.3 vs. 103.9±4.3 μg/dl, p=0.04) and urinary iron excretion (10.4±1.9 vs. 7.4±1.1 μg/24h; p=0.059) were elevated and renal iron deposition attenuated (24.3±1.3 vs. 33.8±2.3; p=0.008) in HbSS VEET KO mice. To further elucidate ET-1/ETA receptor-mediated renal iron accumulation already observed with pharmacological and genetic approaches in HbSS mice, using PEPCK Cre we generated PT specific ETA receptor knockout (PT ETA KO) mice. At baseline, 16 weeks old male and female PT ETA KO mice presented with higher plasma iron concentration (1.0±0.2 vs. 0.5±0.1 μg/ml; p=0.03) and urinary iron excretion (4.5±0.7 vs. 1.7±0.5 μg/24h; p=0.002), whereas renal iron deposition remained unchanged (2.26±0.48 vs. 2.44±0.73 Kpix/ μm; p=0.85). To induce hemolysis with acute iron overload, mice were injected with phenylhydrazine (PhZ, 40 mg/kg, IP; 2 consecutive days) or saline. PhZ significantly reduced hemoglobin in both PT ETA KO and littermate controls when compared to saline injected mice. Moreover, PT ETA KO mice given PhZ had increased plasma iron concentration (4.4±0.3 vs. 3.0±0.5 μg/ml; p=0.03) and urinary iron excretion (5.6±0.7 vs. 3.4±0.7 μg/24h; p=0.05) and decreased renal iron accumulation (0.50±0.22 vs. 0.90±0.20; p=0.011) when compared with saline injected KO control mice. Together, these data demonstrate that ET-1, via ETA receptor activity, contributes to renal iron accumulation in murine models of iron overload.