Islet β cells are the exclusive cellular source of insulin synthesis and secretion, playing a crucial role in regulating blood glucose homeostasis. The impairment of islet β cells due to sustained anaerobic glycolysis pressure represents a significant factor contributing to insulin deficiency or resistance in patients with type 2 diabetes mellitus (T2DM). Glucokinase (GK), serving as the principal rate-limiting step in glucose transport, exhibits close association with insulin secretion. Hypoxia-inducible factor 1α subunit (HIF-1α) and 6-phosphofructo-2-kinase (PFKFB3) act as pivotal regulatory factors within glycolysis. The HIF-1α/PFKFB3 signaling pathway participates in intricate pathophysiological processes involving islet β cell function and maintenance under high-glucose conditions. This review elucidates the mechanisms by which the HIF-1α/PFKFB3 signaling pathway mediates GK induction through anaerobic glycolysis, mitochondrial network adaptive fragmentation, oxidative stress, and dedifferentiation leading to pancreatic islet β cell dysfunction in T2DM, thereby offering novel insights for clinical prevention and treatment strategies targeting T2DM.