The enzyme 6-phosphofructo-1-kinase (PFK) is the main enzyme controlling the glycolytic flux, been regulated by many positive and negative allosteric modulators such as fructose 2,6 biphosphate, AMP been positives and citrate, ATP, and acid pH as negatives modulators. Skeletal muscle is able to produce ATP at very low oxygen supply through anaerobic glycolysis, which produces lactate as final metabolite of the pathway. Here, we evaluated lactate as a modulator of PFK as well as its effects on enzyme tertiary structure. Our results shows that lactate promote a dose-dependent inhibition of muscle PFK within the physiological range of the metabolite, without alterations on the pH of the medium. This inhibition is attenuated by raising the ATP concentration from 1 to 3 mM or the enzyme concentration from 5 to 10, 15 or 20 mg/ml. Actually, the maximal inhibition observed (80 %) with 10 mM lactate, 1 mM ATP and 5 mg/ml PFK was completely abolished when the assay was performed in the presence of 3 mM ATP as well as 15 or 20 mg/ml PFK. These data suggested that the effects of lactate on PFK activity could be due to the dimmerization of the enzyme, which is inhibitory to the enzyme and counteracted by raising the enzyme concentration. However, lactate did not alter the center of mass of instrinsic fluorescence spectra of the enzyme. This data support evidences that lactate is not altering the equilibrium between PFK oligomeric conformers, nor the tertiary structure of the enzyme. Based on these results, we might conclude that lactate is modulating PFK activity in a specific allosteric manner without major alterations on enzyme tertiary or quaternary structure.