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Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme and an attractive target for type 2 diabetes mellitus (T2DM); however, clinical progress has been limited by known inhibitor binding sites. Here, we report the cocrystal structure of human FBPase (HuFBPase) bound to a sulfonylurea-based inhibitor (compound 5, IC₅₀ = 2.93 μM) at a previously unrecognized cryptic allosteric pocket. Compound 5 engages this site via π-π stacking with Y57 and extensive hydrogen-bond interactions, induces conformational rearrangement of K72 and D74, and facilitates the formation of a hydrogen-bond network with S123 that disrupts substrate catalysis. Structure-guided optimization yielded compound 29 with improved potency (IC₅₀ = 0.75 μM). Cellular target engagement was confirmed by thermal stabilization of FBPase in LO2 cells. Notably, compound 29 exhibited hypoglycemic activity in vivo, providing the first evidence that targeting this pocket confers therapeutic benefit. Collectively, our findings establish a novel, druggable allosteric site on HuFBPase for T2DM treatment.