The presence of N-methyl-d-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca²⁺uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca²⁺accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca²⁺homeostasis and modulate oxygen delivery to peripheral tissues.