Oxidized albumin is a reliable marker of oxidative stress in hemodialysis (HD) patients. However, oxidized albumin in vivo and its possible clinical significance has been rarely investigated. In the present study, the qualitative modification of albumin in HD patients (n = 20) was examined and their results were compared with healthy age-matched controls (n = 10). The increase in plasma protein carbonyl levels in HD patients was largely due to an increase in oxidized albumin. Human serum albumin (HSA) of HD patients, HSA of HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. Spectroscopic analysis confirmed that the HD-HSA samples contained higher levels of carbonyls than Normal-HSA. An HPLC analysis also suggested that the state of the purified HSA used throughout the experiments accurately reflects the redox state of albumin in blood. HD-HSA was found to have a decreased the antioxidant activity, and was able to trigger the oxidative burst of human neutrophils, compared to Normal-HSA. HD-HSA was conformationally altered, with its hydrophobic regions more exposed and to have a negative charge. In binding experiments, HD-HSA showed impaired Site II-ligand binding capabilities. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HD patients.