Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3beta (GSK-3beta) and that the activated GSK-3beta destabilizes alphaNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of alphaNAC and ER stress responses involving PERK, ATF4, gamma-taxilin, elF2alpha, Bip, and CHOP. Depletion of alphaNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3beta, and that GSK-3beta inhibition prevented alphaNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, alphaNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that alphaNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3beta may participate upstream in this mechanism.