The regulatory roles and clinical significance of glycolysis in tumor development are extensively reviewed in this article. Glycolysis, a metabolic pathway that converts glucose into lactate under anaerobic conditions, plays a crucial role in tumor growth and progression. Enhanced glycolysis levels, abnormal expression of glycolytic enzymes, and accumulation of glycolytic products are associated with tumor cell proliferation, invasion, metastasis, and immune evasion. Key glycolytic enzymes such as hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase M2 (PKM2), and lactate dehydrogenase (LDH) are highlighted for their roles in these processes. For instance, HK2 and PKM2 promote cell cycle progression, while ENO1 and LDHA contribute to angiogenesis and immune escape. Additionally, glycolysis regulates the tumor microenvironment (TME) by altering pH and promoting angiogenesis and immune cell suppression. The article also discusses the clinical applications of glycolysis in tumor diagnosis and treatment, including the use of glucose analogues like 2-deoxy-D-glucose (2-DG) in positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). Finally, the article reviews novel drugs targeting glycolytic enzymes, such as inhibitors of GLUT1, HK2, and PKM2, which show promise in enhancing the efficacy of anti-tumor therapies.The regulatory roles and clinical significance of glycolysis in tumor development are extensively reviewed in this article. Glycolysis, a metabolic pathway that converts glucose into lactate under anaerobic conditions, plays a crucial role in tumor growth and progression. Enhanced glycolysis levels, abnormal expression of glycolytic enzymes, and accumulation of glycolytic products are associated with tumor cell proliferation, invasion, metastasis, and immune evasion. Key glycolytic enzymes such as hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase M2 (PKM2), and lactate dehydrogenase (LDH) are highlighted for their roles in these processes. For instance, HK2 and PKM2 promote cell cycle progression, while ENO1 and LDHA contribute to angiogenesis and immune escape. Additionally, glycolysis regulates the tumor microenvironment (TME) by altering pH and promoting angiogenesis and immune cell suppression. The article also discusses the clinical applications of glycolysis in tumor diagnosis and treatment, including the use of glucose analogues like 2-deoxy-D-glucose (2-DG) in positron emission tomography (PET) and magnetic resonance spectroscopy (MRS). Finally, the article reviews novel drugs targeting glycolytic enzymes, such as inhibitors of GLUT1, HK2, and PKM2, which show promise in enhancing the efficacy of anti-tumor therapies.