Inhibition of glycolysis at the phosphofructo-1-kinase step slows cell growth. For this reason, overexpression of fructose-2,6-bisphosphatase is a potential target for antineoplasic treatments. However, therapeutic objectives may be compromised by side effects of glycolysis restriction, including enhanced resistance to oxidants and tumor necrosis factor-alpha (TNF-alpha), as well as increased activity of the nuclear factor kappa B (NF-kappaB). In this study we evaluated aspirin as an adjuvant drug for glycolysis restriction by overexpression of fructose-2,6-bisphosphatase. The effect of aspirin on antioxidant defences and NF-kappaB activity were evaluated both in control cells and in fructose-2,6-bisphosphatase-overexpressing cells. Interestingly, aspirin-induced inhibition of NF-kappaB activity was greater in transfectants with restricted glycolysis than in control cells. Our results indicate that aspirin is a suitable complement to therapy based on glycolysis restriction to overcome resistance associated with increased NF-kappaB activity and oxidative stress.