We investigated cellular injury and death induced by ultrapure human Hb (HbA₀) and its diaspirin cross-linked derivative DBBF-Hb in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to hydrogen peroxide (H₂O₂). HbA₀ underwent extensive degradation and heme loss, whereas DBBF-Hb persisted longer in its ferryl (Fe⁴⁺) form. The formation of ferryl HbA₀ or ferryl DBBF-Hb was associated with a significant decrease in endothelial cell GSH compared with the addition of H₂O₂ or Hbs alone. This effect was inhibited by catalase, but not by superoxide dismutase or deferoxamine mesylate. The presence of HbA₀ and DBBF-Hb reduced H₂O₂-induced apoptosis, as measured by cell morphology, annexin V binding assay, and caspase inhibition, consistent with the ability to consume H₂O₂ in an enzyme-like fashion. However, the pattern of cell death and injury produced by HbA₀ and DBBF-Hb appeared to be distinctly different among proteins as well as among cells with and without GSH. These findings may have important implications for the use of cell-free Hb as oxygen therapeutics in patients with coexisting pathologies who may lack antioxidant protective mechanisms.