The lumen of the endoplasmic reticulum constitutes a separate intracellular compartment with a special proteome and metabolome. The redox conditions of the organelle are also characteristically different from those of the other subcellular compartments. The luminal environment has been considered more oxidizing than the cytosol due to the presence of oxidative protein folding. However, recent observations suggest that redox systems in reduced and oxidized states are present simultaneously. The concerted action of membrane transporters and oxidoreductase enzymes maintains the oxidized state of the thiol–disulfide and the reduced state of the pyridine nucleotide redox systems, which are prerequisites for the normal redox reactions localized in the organelle. The powerful thiol-oxidizing machinery of oxidative protein folding continuously challenges the local antioxidant defense. Alterations of the luminal redox conditions, either in oxidizing or reducing direction, affect protein processing, are sensed by the accumulation of misfolded/unfolded proteins, and may induce endoplasmic reticulum stress and unfolded protein response. The activated signaling pathways attempt to restore the balance between protein loading and processing and induce programmed cell death if these attempts fail. Recent findings strongly support the involvement of redox-based endoplasmic reticulum stress in a plethora of human diseases, either as causative agents or as complications. Antioxid. Redox Signal. 13, 77–108