Potentilla discolor is used as an ethnomedicine in treatments of diabetes mellitus in China for years. In the present study, the anti-hyperglycemic effects of a clinical active extract (decoction) from P. discolor were investigated in Ob-db mice. Four week's treatment of P. discolor decoction ameliorated the development of hyperlipidemia, lipid peroxidation and hyperglycemia associated with hyperphagia and polydypsia in Ob-db mice. P. discolor significantly attenuated the increase of blood glucose and cholesterol levels in Ob-db mice. These findings clearly provided evidences regarding the anti-hyperglycemic potentials of P. discolor decoction. High-resolution liquid chromatography-mass spectrometry/mass spectrometry (HR-LC-MS/MS) was used to analyze the phytochemicals in P. discolor decoction. In an comprehensive analysis of phytochemicals in P. discolor, thirty-five components were identified or characterized in P. discolor decoction and only sixteen of them have been reported in P. discolor previously. There are five major components identified in P. discolor decoction. One of the major components is a flavonoid sulfate, and this is the first evidence for the presences of sulfated flavonoid in P. discolor. Sulfated flavonoids have been reported to improve the complications of diabetes mellitus by inhibition of the aldose reductase in both experimental animals and clinical trials. Therefore, the sulfated flavonoid in P. discolor decoction may in part contribute to the anti-hyperglycemic effect of P. discolor.