A phytochemical investigation of the roots of Euphorbia fischeriana Steud. led to the isolation of eleven undescribed gallotannins, fishertannins A-K, together with four known analogues. Their structures were elucidated by the comprehensive spectroscopic data including UV, IR, HR-ESI-MS, and NMR, while the absolute configurations of the sugar moiety were determined by the acid hydrolysis and HPLC analyses. Fishertannin A possessed an unusual skeleton comprised of acetophenone, galloyl group, arabinofuranosyl and glucopyranosyl moieties. Fishertannin B, fishertannin H, fishertannin K, 1,2,3-tri-O-galloyl-β-D-glucopyranose, 3,4,6-tri-O-galloyl-D-glucopyranose, and 1,6-di-O-galloyl-β-D-glucopyranose displayed the potent α-glucosidase inhibitory activities with the IC50 values of 15.48-177.13 μM. Examination of the structure-activity relationships (SAR) demonstrated that the galloyl and glucopyranosyl moieties played a key role in the inhibitory activity for both α-glucosidase and α-amylase inhibitory activity. Among all isolates, 1,2,3-tri-O-galloyl-β-D-glucopyranose showed the most potent and highly specific inhibitory activity against α-glucosidase with an IC50 value of 15.48 ± 0.60 μM. The kinetic analysis of 1,2,3-tri-O-galloyl-β-D-glucopyranose disclosed the mixed inhibition type on α-glucosidase, and the molecular docking visualized the stable binding with the catalytic pocket of α-glucosidase (pdb 3A4A). These findings indicated the excellent antidiabetic potential of the gallotannins from E. fischeriana, while 1,2,3-tri-O-galloyl-β-D-glucopyranose could be developed as a promising candidate for the treatment of T2DM with fewer side effects.Copyright © 2022 Elsevier Ltd. All rights reserved.