Glycolysis-depleted cells, obtained by stable transfection of fructose 2,6-bisphosphatase in mink lung epithelial cells (Mv1Lu), were less sensitive to serum withdrawal- and TNF-alpha-induced apoptosis than cells transfected with the empty vector pcDNA3 (control cells). We compared the differences in the redox status of the two transfectants and the changes produced by TNF-alpha treatment. The activities of the antioxidant enzymes catalase and glutathione peroxidase, as well as the content of reduced glutathione (GSH) and the activity of the nuclear transcription factor kappa B (NF-kappa B), were higher in pFBPase-2 clones than in control cells in all the conditions tested. TNF-alpha challenge sharpened the differences in glutathione peroxidase activity, GSH/GSSG ratios, and NF-kappa B activation between transfectants. These data indicate that glycolysis restriction at the PFK step protects cells against apoptotic stimuli by increasing the GSH content and NF-kappa B activity. This acquired feature may compromise antineoplastic treatments based on glycolytic depletion.