6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway.