Breast cancer cells undergo a process of reprogramming their metabolism for rapid growth and proliferation. One of the most common metabolic changes is aerobic glycolysis (Warburg effect), which leads to increased lactate generation and glucose uptake capacity. Triosephosphate isomerase (TPI) is a key enzyme in glycolysis. The effect of Resveratrol (RES), a natural plant compound with known anti-cancer properties, on the TPI enzyme is unknown. The purpose of this study is to examine how RES relates to TPI in breast cancer. TPI levels were examined by ELISA and western-blotting methods in MCF-7 and MDA-MB-231 cells. The changes in lactate dehydrogenase (LDH) activity, methylglyoxal (MGO) formation, nitric oxide synthase (eNOS and iNOS) levels, and MAPK signaling pathway were investigated by colorimetric assays and western-blotting. It was shown for the first time that RES induced a significant decrease in TPI in a dose-dependent manner, with a concomitant increase in levels of MGO, a toxic intermediate. Furthermore, RES treatment decreased LDH activity, and the expression of MAPK, ERK1/2, and JNK, while increasing the expression of eNOS and iNOS levels. The results sign a potential cytotoxic effect of RES due to increased MGO levels resulting from TPI inhibition. The effect of RES on TPI function and glycolysis may be related to NOS induction and the MAPK pathway. These findings are the first data showing the effect of RES treatment on TPI, suggesting that TPI may be a target for energy metabolism in breast cancer.