Fructose-1,6-bisphosphatase (FBPase), as a key rate-limiting enzyme in the gluconeogenesis (GNG) pathway, represents a practical therapeutic strategy for type 2 diabetes (T2D). Our previous work first identified cysteine residue 128 (C128) was an important allosteric site in the structure of FBPase, while pharmacologically targeting C128 attenuated the catalytic ability of FBPase. Herein, ten approved cysteine covalent drugs were selected for exploring FBPase inhibitory activities, and the alcohol deterrent disulfiram displayed superior inhibitory efficacy among those drugs. Based on the structure of lead compound disulfiram, 58 disulfide-derived compounds were designed and synthesized for investigating FBPase inhibitory activities. Optimal compound 3a exhibited significant FBPase inhibition and glucose-lowering efficacy in vitro and in vivo. Furthermore, 3a covalently modified the C128 site, and then regulated the N125–S124–S123 allosteric pathway of FBPase in mechanism. In summary, 3a has the potential to be a novel FBPase inhibitor for T2D therapy. Display omitted •Alcohol deterrent disulfiram was repurposed as FBPase covalent inhibitor.•Derivative 3a showed potent FBPase inhibition activity both in vitro and in vivo.•Derivative 3a could ameliorate glucose tolerance both in ICR mice and diabetic db/db mice.•Derivative 3a covalently modified the C128 site of FBPase and affected the catalytic activity of FBPase.