Fisetin (3,3′,4′,7-tetrahydroxyflavone) has been found to be neuroprotective, induce neuronal differentiation, enhance memory, and inhibit the aggregation of the amyloid β protein (Aβ) that may cause the progressive neuronal loss in Alzheimer's disease. The diverse collection of biological activities of this compound may lead to a new type of therapeutic drug for Alzheimer's disease. As the first step to design even more effective drugs based upon the structure of fisetin, the present study investigated the structural requirements for the anti-amyloidogenic activity of fisetin by comparing the effects of several structurally related flavonoids on Aβ fibril formation in vitro. Aβ1-42 (20μM) and the flavonoids were incubated for 0–48h at 37°C, and fibril formation was quantitatively determined by the thioflavin T fluorescence assay. Among ten flavonoids tested, fisetin, 3′,4′,7-trihydroxylflavone, 3,3′,4′-trihydroxyflavone, luteolin, quercetin and myricetin inhibited Aβ fibril formation. On the other hand, 3,3′,7-trihydroxyflavone, 5-deoxykaempferol, chrysin and kaempferol enhanced Aβ fibril formation. These results suggest that the 3′,4′-dihydroxyl group, but not the 3- or 7-hydroxyl group, is essential for the inhibitory effect of fisetin on Aβ fibril formation.