Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased 59Fe uptake from transferrin by 1.5–2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased 59Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated 59Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of 59Fe uptake from 59Fe-citrate, only intracellular ascorbate is needed for transferrin 59Fe uptake. Additionally, experiments with l-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent 59Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or 125I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.