Flavonoids are ubiquitous components in vegetables, fruits, tea, and wine. Therefore, they are often consumed in large quantities in our daily diet. Several flavonoids have been shown to have potential as antidiabetic agents. In the present study, we focused on inhibition of glycogen phosphorylase (GP) by flavonoids. 6-Hydroxyluteolin, hypolaetin, and quercetagetin were identified as good inhibitors of dephosphorylated GP (GPb), with IC50 values of 11.6, 15.7, and 9.7 μM, respectively. Furthermore, a structure-activity relationship study revealed that the presence of the 3′ and 4′ OH groups in the B-ring and double bonds between C2 and C3 in flavones and flavonols are important factors for enzyme recognition and binding. Quercetagetin inhibited GPb in a noncompetitive manner, with a Ki value of 3.5 μM. Multiple inhibition studies by Dixon plots suggested that quercetagetin binds to the allosteric site. In primary cultured rat hepatocytes, quercetagetin and quercetin suppressed glucagonstimulated glycogenolysis, with IC50 values of 66.2 and 68.7 μM, respectively. These results suggested that as a group of novel GP inhibitors, flavonoids have potential to contribute to the protection or improvement of control of diabetes type II.