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Clotrimazole is an antifungal azole derivative recently recognized as a calmodulin antagonist with promising anticancer effects. This property has been correlated with the ability of the drug to decrease the viability of tumor cells by inhibiting their glycolytic flux and consequently decreasing the intracellular concentration of ATP. The effects of clotrimazole on cell glycolysis and ATP production are considered to be due to the detachment of the glycolytic enzymes from the cytoskeleton. Here, we show that clotrimazole directly inhibits the key glycolytic enzyme 6-phosphofructo-1-kinase (PFK). This property is independent of the anti-calmodulin activity of the drug, since it is not mimicked by the classical calmodulin antagonist compound 48/80. However, the clotrimazole-inhibited enzyme can be activated by calmodulin, even though calmodulin has no effect on PFK activity in the absence of the drug. Clotrimazole alone induces the dimerization of PFK reducing the population of tetramers, which is not observed when calmodulin is also present. Since PFK dimers are less active than PFK tetramers, this can explain the inhibitory effect of clotrimazole on the enzyme. Additionally, clotrimazole positively modulates the association of PFK with erythrocyte membranes. Altogether, our data support a hitherto unrecognized action of clotrimazole as a negative modulator of glycolytic flux through direct inhibition of the key enzyme PFK.