The multifunctional cytoprotective protein peroxiredoxin 6 (Prdx6) maintains cellular homeostasis and membrane integrity by regulating expression of intracellular reactive oxygen species (ROS) and phospholipid turnover. Using cells derived from targeted inactivation of Prdx6 gene or its depletion by RNA interference or aging, we showed that Prdx6 deficiency in cells evoked unfolded protein response (UPR), evidenced by increased expression or activation of proapoptotic factors, CHOP, ATF4, PERK, IRE-α and eIF2-α and by increased caspases 3 and 12 processing. Those cells displayed enhanced and sustained expression of endoplasmic reticulum (ER) stress-related chaperon proteins, Bip/glucose-regulated protein 78, calnexin, and calreticulin. Under cellular stress induced by hypoxia (1% O(2) or CoCl(2) treatment) or tunicamycin, Prdx6-deficient cells exhibited aberrant activation of ER stress-responsive genes/protein with higher expression of ROS, and died with apoptosis. Wild-type cells exposed to tunicamycin or hypoxia remained relatively insensitive with lower expression of ROS and ER-responsive genes than did Prdx6-deficient cells, but upregulation of ER stress responsive proteins or chaperones mimicked the UPR response of Prdx6-deficient or aging cells. Expression of Prdx6 blocked ER stress-induced deleterious signaling by optimizing physiologically aberrant expression of ER stress responsive genes/proteins in Prdx6-deficient cells or cells facing stressors, and rescued the cells from apoptosis. These findings demonstrate that impaired homeostasis and progression of pathogenesis in Prdx6-deficient lens epithelial cells or in aging cells should be blocked by a supply of Prdx6. The results provide a new molecular basis for understanding the etiology of several age-associated degenerative disorders, and potentially for developing antioxidant Prdx6-based therapeutics.