AIM: To screen a potential PTP1b inhibitor from the microbial origin-based compound library and to investigate the potential anti-diabetic effects of the inhibitor in vivo and determine its primary anti-diabetic mechanism in vitro and in silico. METHODS: PTP1b inhibitory activity was measured using recombination protein as the enzyme and p-NPP as the substrate. The binding of the inhibitor to PTP1b was analysed by docking in silico and confirmed by ITC experiments. The intracellular signalling pathway was detected by Western blot analysis in HepG2 cells. The anti-diabetic effects were evaluated using a diabetic mice model in vivo. RESULTS: Among 545 microbial origin-based pure compounds tested, trivaric acid, a tridepside, was selected as a PTP1B inhibitor exhibiting strong inhibitory activity with an IC of 173nM. Docking and ITC studies showed that trivaric acid was able to spontaneously bind to PTP1b and may inhibit PTP1b by blocking the catalytic domain of the phosphatase. Trivaric acid also enhanced the ability of insulin to stimulate the IR/IRS/Akt/GLUT2 pathway and increase the glucose consumption in HepG2 cells. In diabetic mice, trivaric acid that had been encapsulated into Eudrgit L100-5.5 showed significant anti-diabetic effects, improving insulin resistance, leptin resistance and lipid profile and weight control at doses of 5mg/kg and 50mg/kg. SIGNIFICANCE: Trivaric acid is a potential lead compound in the search for anti-diabetic agents targeting PTP1b.