Cancer cells have evolved metabolic mechanisms to enable rapid proliferation, survival, and metastasis. Among these mechanisms, glycolysis and non-glycolytic acid metabolism perform vital functions. The glycolytic enzyme glucose-6-phosphate isomerase (G6PI) promotes the simultaneous conversion of glucose-6-phosphate as well as fructose-6-phosphate, promoting glycolysis and gluconeogenesis; it functions as both a metabolic regulator and an external signal molecule. Extracellularly, GPI functions as an autocrine motility factor (AMF), facilitating tumor cell movement, invasion, and metastasis. GPI functions intracellularly to regulate redox equilibrium, promote anabolic pathways, and influence gene expression linked to cancer survival. GPI’s multiple activities also allow cancer cells to withstand pharmacological stresses, contributing to resistance to both chemotherapy and radiotherapy via mechanisms such as increased DNA repair, elevated survivability pathways, and metabolic adaptability. As an effective target, GPI provides an exceptional chance for intervention. Small-molecule inhibitors of its enzymatic activity have demonstrated efficacy in preclinical investigations, inhibiting cancer cell growth and triggering apoptosis. Strategies that integrate GPI inhibition with additional medications, such as immune checkpoint inhibitors or metabolic modulators, may improve the effectiveness of therapy and overcome resistance. This chapter discusses the interactions between GPI’s glycolytic and non-glycolytic structures, their functions, regulation, and role in cancer progression and metastasis, as well as targeting G6PI as a cancer therapy method.