Despite evidence suggesting a significant role of pyruvate kinase muscle isozyme (PKM) in cancer development, its particular function in colorectal cancer (CRC) remains unclear. This study aimed to elucidate the specific role and mechanism of PKM and its isoforms, PKM1 and PKM2, in the progression of CRC.We analyzed PKM, PKM1, and PKM2 expression in CRC tissues and their correlation with clinicopathological features. Plasmids were constructed to modulate these isoforms' expression in CRC cells. Cellular behavior changes, including glucose metabolism alterations, were assessed using the Seahorse Energy Meter, and the Cell Counting Kit-8 (CCK8) assay to determine the inhibitory concentration of 5-fluorouracil (5-FU) on different CRC cell groups.Our results showed significant PKM overexpression in CRC tissues, which was correlated with negative prognostic factors such as advanced T stages and lymph node metastasis. A lower PKM1/PKM2 ratio was associated with these adverse outcomes. Functionally, PKM1 overexpression decreased cell migration and invasion, increasing 5-FU sensitivity. Conversely, PKM2 overexpression promoted malignant traits and reduced 5-FU sensitivity. Intriguingly, the introduction of glycolysis inhibitors attenuated the impact of PKM on the biological functions of CRC cells, suggesting a glycolysis-dependent mechanism.This study establishes the PKM1/PKM2 ratio as crucial in CRC progression and 5-FU response. PKM1 overexpression reduces CRC malignancy and increases 5-FU sensitivity, while PKM2 does the opposite. Notably, glycolysis inhibitors lessen PKM's impact on CRC cells, highlighting a glycolysis-dependent mechanism. These insights suggest targeting PKM isoforms and glycolysis pathways as a promising CRC therapeutic strategy, potentially enhancing treatment efficacy.2024 Translational Cancer Research. All rights reserved.