Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that participates in various cellular events, such as DNA repair and apoptosis. The functional diversity of GAPDH depends on its intracellular localization. Because AMP-activated protein kinase (AMPK) regulates the nuclear translocation of GAPDH in young cells and AMPK activity significantly increases during aging, we investigated whether altered AMPK activity is involved in the nuclear localization of GAPDH in senescent cells. Age-dependent nuclear translocation of GAPDH was confirmed by confocal laser scanning microscopy in human diploid fibroblasts (HDFs) and by immunohistochemical analysis in aged rat skin cells. Senescence-induced nuclear localization was reversed by lysophosphatidic acid but not by platelet-derived growth factor. The extracellular matrix from young cells also induced the nuclear export of GAPDH in senescent HDFs. An activator of AMPK, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), increased the level of nuclear GAPDH, whereas an inhibitor of AMPK, Compound C, decreased the level of nuclear GAPDH in senescent HDFs. Transfection with AMPKα siRNA prevented nuclear translocation of GAPDH in senescent HDFs. The stimulatory effect of AICAR and serum depletion on GAPDH nuclear translocation was reduced in AMPKα1/α2-knockout mouse embryonic fibroblasts. Overall, increased AMPK activity may play a role in the senescence-associated nuclear translocation of GAPDH.