End-stage renal disease (ESRD) is associated with a variety of erythron alterations including structural and functional changes in circulating erythrocytes (RBCs), which are potential risk factors to cause RBC malfunctions and corresponding anemia of different severity. However, the mechanisms of such changes in RBCs remain understudied. Here we used flow cytometry to estimate intracellular esterase activity, phosphatidylserine externalization, and reticulocyte count and state, laser diffraction for osmotic and ammonium stress tests, and spectrophotometry to evaluate the generation of unstable hemoglobin (Hb) forms in RBCs of patients with ESRD before and after the hemodialysis (HD) session. RBCs in ESRD were more osmotically fragile and had an increased swelling rate in the ammonium stress test. HD did not affect RBC deformability but led to Hb oxidation to ferryl forms and triggered such apoptosis-like events, as a decrease in intracellular esterase activity and an increase in the number of annexin-V-positive cells. Our data indicate that uremic syndrome, combined with the mechanical and chemical effects of HD therapy, challenges the erythron of HD patients and contributes to a multifactorial decrease in RBC function and aggravation of renal anemia.