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Recent research has revealed that the heightened glycolysis levels, the abnormal expression of glycolytic enzymes, and the accumulation of glycolytic products could regulate the growth, proliferation, invasion, and metastasis of tumor cells and provide a favorable microenvironment for tumor development and progression. Based on the distinctive glycolytic characteristics of tumor cells, novel imaging tests have been developed to evaluate tumor proliferation and metastasis. In addition, glycolytic enzymes have been found to serve as promising biomarkers in tumor, which could provide assistance in the early diagnosis and prognostic assessment of tumor patients. Numerous glycolytic enzymes have been identified as potential therapeutic targets for tumor treatment, and various small molecule inhibitors targeting glycolytic enzymes have been developed to inhibit tumor development and some of them are already applied in the clinic. 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