Acetylsalicylic acid (ASA) is one of the most used drugs in world and its consumption exceeds 20.000 tons/year. It has been known for more than a century that salicylates are able to reduce fasting blood glucose in diabetic patients. The cellular and molecular mechanism of the hypoglycemic activity of aspirin has not been well elucidated. For the mechanism of salicylates activity, several hypotheses were raised 20 years ago explaining the hypoglycemic action. The extra-pancreatic hypothesis, which suggests salicylates action through a pancreatic-independent mechanism, seems to have become dominant as it has gained substantial support from studies published recently. This hypothesis proposes that the effects of salicylates might be directly in cell metabolism and not on normal hormone levels. Cellular glucose metabolism occurs through the glycolytic pathway where 6-phosphofructo-1-kinase (PFK) plays a crucial role on its regulation. The aim of this study is to evaluate the effects of ASA on PFK activity since modulation of the enzyme can reflect on the pathway flux. We show that ASA promote a dose-dependent inhibition of PFK which is not irreversible and the inhibition kinetics shows a Ki of 6.6 mM for ASA. It shows an negative cooperativity indicating that low concentrations of ASA promote a more significant effect on PFK activity. Additionally, the inhibition of PFK promoted by ASA is modulated by enzyme concentration suggesting that the observed effects might be due to allosteric binding of the compound which can affect the oligomeric equilibrium of the enzyme. In conclusion, the results reported here can be correlated with the effects of ASA on the glycemia, since it clearly modulates PFK activity and consequently the glycolytic pathway.