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Rheum tanguticum Maxim. ex Balf. (RT), mainly is mainly produced in Gansu, Qinghai, Tibet and Sichuan of China, has long been used as a medicine for purging fire and quenching thirst. The RTR-2 (Rheum tanguticum root extract without polysaccharides and protein) had strong inhibitory activities against a -amylase, maltase and sucrase with the IC 50 0.042 mg/mL, 0.124 mg/mL and 0.097 mg/mL, respectively. In addition, RTR-2 could significantly reduce PBG level of diabetic mice in starch/maltose/sucrose tolerance test. Total 48 constituents including 12 anthraquinones, 4 bianthrones, 8 flavans, 3 stilbenes, 5 phenylbutanones, 4 naphthosides, 8 glucose gallates and 4 phenolic acids were determined in RTR-2 by UPLC-Triple-TOF/MS analysis of molecular ion, main fragments, retention time, and references. Furthermore, the components such as flavan ((-)-epicatechin 3- O -gallate) and phenylbutanone (lindleyin) were demonstrated to have sucrase and maltase inhibitory activites, and might be the main hypoglycemic active ingredients of RTR-2 extract. All these results offered an exhaustive knowledge for the hypoglycemic compounds of Rheum tanguticum , which was conducive to its further development and utilization as a valuable source of hypoglycemic plant in pharmaceutical industry. [Display omitted] • Firstly demonstrated anti-hyperglycaemic effects of RT root in vivo and in vitro. • 48 ingredients in RT were detected and identified by UPLC-Triple-TOF-MS. • Analyzed interrelationships between activity assay and ingredients. [ABSTRACT FROM AUTHOR]