Kidney disease, including proximal tubule (PT) dysfunction, and Vitamin D deficiency are among the most prevalent complications in sickle cell disease (SCD) patients. Although these two comorbidities have never been linked in SCD, the PT is the primary site for activation of Vitamin D. Precursor 25-hydroxyVitamin D [25(OH)D] bound to Vitamin D-binding protein (DBP) is taken up by PT cells via megalin/cubilin receptors, hydroxylated to the active 1,25-dihydroxyVitamin D [1,25(OH)2D] form, and released into the bloodstream. We tested the hypothesis that cell-free hemoglobin (Hb) filtered into the PT lumen impairs Vitamin D uptake and metabolism. Hb at concentrations expected to be chronically present in the ultrafiltrate of SCD patients competed directly with DBP for apical uptake by PT cells. By contrast, uptake of retinol binding protein was impaired only at considerably higher Hb concentrations. Prolonged exposure to Hb led to increased oxidative stress in PT cells and to a selective increase in mRNA levels of the CYP27B1 hydroxylase, although protein levels were unchanged. Hb exposure also impaired Vitamin D metabolism in PT cells, resulting in reduced ratio of 1,25(OH)2D:25(OH)D. Moreover, plasma levels of 1,25(OH)2D were reduced in a mouse model of SCD. Together, our data suggest that Hb released by chronic hemolysis has multiple effects on PT function that contribute to Vitamin D deficiency in SCD patients.