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Neurotrophic factors are playing vital roles in survival, growth, and function of neurons. Regulation of neurotrophic factors in the brain has been considered as one of the targets in developing drug or therapy against neuronal disorders. Flavonoids, a family of multifunctional natural compounds, are well known for their neuronal beneficial effects. Here, the effects of flavonoids on regulating neurotrophic factors were analyzed in cultured rat astrocytes. Astrocyte is a major secreting source of neurotrophic factors in the brain. Thirty-three flavonoids were screened in the cultures, and calycosin, isorhamnetin, luteolin, and genistein were identified to be highly active in inducing the synthesis and secretion of neurotrophic factors, including nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF). The inductions were in time- and dose-dependent manners. In cultured astrocytes, the phosphorylation of estrogen receptor was triggered by application of flavonoids. The phosphorylation was blocked by an inhibitor of estrogen receptor, which in parallel reduced the flavonoid-induced expression of neurotrophic factors. The results proposed the role of flavonoids in protecting brain diseases, and therefore these flavonoids could be developed for health food supplement for patients suffering from neurodegenerative diseases.