As dual regulators, the PTP-1B signaling pathway and α-glucosidase slow glucose release and increase the degree of insulin sensitivity, representing a promising therapeutic target for type 2 diabetes. In this study, we systematically examined the in vivo and in vitro anti-diabetic activities of natural flavonoids 1-6 from Chrysanthemum morifolium. Flavonoids 1-6 increased glucose consumption-promoting activity and the phosphorylation of GSK-3β and Akt, and decreased PTP-1B protein level along with slightly inhibitory activity of the PTP-1B enzyme. Moreover, flavonoids 1-2 treatment induced insulin secretion in INS-1 cells. Besides, in vivo study revealed that flavonoids 2 and 5 demonstrated potent anti-hyperglycemic and anti-hyperlipidemic activity, and improved maltose and glucose tolerance. Although flavonoid 2 exhibited lower inhibitory activity against α-glucosidase in vitro, it could deglycosylated in vivo to diosmetin to function as an α-glucosidase inhibitor. Taken together, these results led to the identification of the natural flavonoids 1-6 from C. morifolium as dual regulators of α-glucosidase and the PTP-1B signaling pathway, suggesting their potential application as new oral anti-diabetic drugs or functional food ingredients.